Derivatives of succinic and glutaric acids and analogs thereof useful as inhibitors of phex

ABSTRACT

The present invention relates to derivatives of succinic and glutaric acids and analogues thereof, having the following general formula: 
     
       
         
         
             
             
         
       
     
     useful as inhibitors of PHEX. These derivatives are useful for promoting generation of bone mass and treating or preventing diseases or conditions associated with a phosphate metabolism defect. Methods for preparation and intermediates are also disclosed.

This application claims the benefit of U.S. Provisional Application No.60/430,382, filed Dec. 3, 2002. The entire text of the above provisionalapplication is specifically incorporated by reference.

BACKGROUND OF THE INVENTION

A. Field of Invention

The present invention relates to derivatives of succinic and glutaricacids and analogs thereof, useful as inhibitors of PHEX. Morespecifically, the present invention relates to derivatives of succinicand glutaric acids and analogs thereof useful for promoting fasterregeneration of bone mass after bone fractures, implantation oforthopedic and dental prostheses, or after loss of bone mass as aconsequence of bone diseases such as osteoporosis.

B. Related Art

The PHEX gene (formerly PEX; Phosphate regulating gene with homologiesto endopeptidases on the X chromosome) was identified by a positionalcloning approach as the candidate gene for X-linked hypophosphatemia(XLH) (The Hyp Consortium, 1995). XLH is a Mendelian disorder ofphosphate homeostasis characterized by growth retardation, rachitic andosteomalacic bone disease, hypophosphatemia, and renal defects inphosphate re-absorption and vitamin D metabolism (Tenenhouse and Econs,2001).

Several groups have cloned and sequenced the human and mouse PHEX/PhexcDNAs (PHEX/Phex refers to the human and mouse genes, respectively) (Duet al., 1996; Lipman et al., 1998; Grieff et al., 1997; Beck et al.,1997; Guo and Quarles, 1997; Strom et al., 1997). Amino acid sequencecomparisons have demonstrated homologies between PHEX/Phex protein andmembers of the neutral endopeptidase family, as previously observed inthe partial sequence of the candidate PHEX gene (The HYP Consortium,1995). The peptidases of the neutral endopeptidase family arezinc-containing type II integral membrane glycoproteins having arelatively short cytoplasmic N-terminal region, a single transmembranedomain, and a long extracytoplasmic domain containing the active site ofthe enzyme (Turner and Tanzawa, 1997).

Much of the present knowledge about XLH has been obtained from studiesof the Hyp mouse, which harbours a large deletion of the Phex gene (Becket al., 1997) and which has been used as an animal model of the humandisease (Tenenhouse, 1999). In particular, these animals show increasedrenal phosphate excretion due to a down-regulation of the Npt2 phosphatetransporter, which is necessary for the re-absorption of phosphate fromthe nephron. The serum concentration of 1,25(OH)₂D3 (calcitriol) wasfound to be the same in Hyp mice as in normal littermates. However, theHyp kidney showed an accelerated degradation of the vitamin D metaboliteto 1,24,25(OH)₃D3, a metabolite with reduced activities. In the presenceof a phosphate rich diet, Hyp mice were shown to experience an increasein serum 1,25(OH)₂D3 and a drop in C-24 oxidation products, in contrastto normal mice which experienced no such changes. The renal disorder invitamin D metabolism in Hyp mice appears to be secondary to thephosphate disorder.

The mechanism by which loss of PHEX function elicits the observed boneand renal abnormalities in XLH patients is not clear. There are no datasuggesting the presence of PHEX/Phex mRNA in the kidney (Du et al.,1996; Beck et al., 1997; Grieff et al., 1997). However, considering thesimilarities between the PHEX protein and the other members of thismetallopeptidase family, it has been speculated that PHEX may regulaterenal phosphate reabsorption by controlling the activity of acirculating factor. It was demonstrated that the inhibition ofNa-dependent phosphate transport in cultured renal cells can be achievedby a factor in conditioned medium from cultured osteoblasts derived fromHyp mice (Lajeunesse et al. 1996; Nesbitt et al., 1999).

Phosphaturic activity(ies) have also been discovered in tumors frompatients with tumor-induced osteomalacia (TIO, also known as oncogenichypophosphatemic osteomalacia), an acquired renal phosphate wastingdisorder with the phenotypic features of XLH (Tenenhouse and Econs,2001). The term “phosphatonin” was designated to depict the phosphaturictumor factor(s) (Econs and Drezner, 1994) and although the exact natureof “phosphatonin” remains to be determined, several candidates have beenproposed (Schiavi and Moe, 2002). It has been shown that mutations inthe FGF-23 gene which encodes a novel growth factor, fibroblast growthfactor-23 (FGF-23), is responsible for Autosomal DominantHypophosphatemic Rickets (ADHR), an inherited disorder that resemblesXLH and TIO (ADHR Consortium, 2001). Moreover, it has been demonstratedthat overexpression of FGF-23 in animal models elicits renal phosphatewasting, a reduction in serum phosphate levels and osteomalacia (Shimadaet al., 2001). Of interest are the findings that FGF-23 is overexpressedin tumors from patients with TIO (Jan de Beur et al., 2002) as well asin XLH patients (Jonsson et al., 2002). In addition to FGF-23, otherproteins such as Frizzled-related protein 4 (FRP-4) (Jan de Beur et al.,2002) and MEPE (matrix extracellular phosphoglycoprotein) (Rowe et al.,2000) are overexpressed in TIO tumors.

PHEX/Phex mRNA has been detected in bones by Northern blot hybridizationand in other adult and fetal tissues such as lungs, liver, muscles, andovaries by RT-PCR and RNase protection assays (Du et al., 1996; Beck etal., 1997). In situ hybridization performed on sections of embryos andnewborn mice showed the presence of Phex mRNA in osteoblasts andodontoblasts (Ruchon et al., 1998). Phex gene expression was detectableon day 15 of embryonic development, which coincides with the beginningof intracellular matrix deposition in bones. Moreover, Northern blottinganalysis of total RNA from calvariae and from teeth of 3-day-old andadult mice showed that the abundance of the Phex transcript haddecreased in adult bones and in non growing teeth. This result wasconfirmed when the presence of the Phex protein in newborn adult boneswas investigated by Western blotting using a monoclonal antibody raisedagainst the human PHEX. Immunohistochemical studies on a 2 month-oldmouse showed exclusive labeling of mature osteoblasts and osteocytes inbones, and of odontoblasts in teeth (Ruchon et al., 2000). These resultssuggest that PHEX/Phex is important in both the development andmaintenance of mineralization in these tissues. This hypothesis wassupported by evidence of intrinsic abnormalities in osteoblasts from Hypmice (Ecarot et al., 1992; Karaplis 2003). PHEX might thus be involvedin the control of bone metabolism, both indirectly at the kidney levelby controlling renal phosphate reabsorption, and directly at the bonelevel by inactivating a trophic peptide factor controlling eitherosteoblast or osteoclast functions or both.

Osteogenesis is a complex biological process that includes proliferationand differentiation of bone-forming cells (osteoblasts), synthesis of anorganic matrix composed mainly of type I collagen, and mineralization ofthe organic matrix by deposition of hydroxyapatite crystals. Varioustechnologies have been developed to stimulate osteogenesis for boneregeneration in osseous reconstructive surgery. These include the use ofbone morphogenetic proteins (BMPs) as osteogenic agents, mostly incombination with a solid support such as metal meshes (Vehof et al.,2001), atelopeptide type I collagen (Ikeuchi et al., 2002) orhydroxyapatite (Yoshida et al., 1999). Hydroxyapatite is anosteoconductive material that maintains an original biocompatible form.During reconstruction of bone defects, its osteoconduction can beenhanced with osteogenic agents such as BMPs. More recently, evidencehas emerged that novel and still poorly characterized peptides mightalso be useful for stimulating osteogenesis. These peptides are thoughtto be involved in poorly characterized pathways regulating bonemineralization. It is surmised that these pathways could be under thecontrol of PHEX.

There thus remains a need to develop selective PHEX inhibitors tocontrol phosphate metabolism and which can be used as osteogenic agents,as well as methods of administering the PHEX inhibitors.

The present invention seeks to meet these and other needs. The presentdescription refers to a number of documents, the contents of which isherein incorporated by reference in their entirety.

SUMMARY OF THE INVENTION

There are many reports in the patent and scientific literature ofcompounds useful as inhibitors of zinc metallopeptidases. Morespecifically, the compounds of relevance in such reports are based onstructures comprising a zinc ligand such as a thiolate, carboxylate,hydroxamate, phosphonate or phosphinate, linked directly or tethered tothe N-terminal side of a dipeptide or analog thereof. An additionalfeature of these compounds is the characteristic presence of ahydrophobic side chain on the N-terminal amino acid or analog thereofbearing the zinc ligand. These compounds are useful mainly as ACE, NEP,ACE-NEP, ECE and MMP inhibitors (U.S. Pat. No. 4,337,201; U.S. Pat. No.5,362,727; U.S. Pat. No. 5,380,921; Roques et al., 1993; Whittaker, etal., 1999). As disclosed in the present invention, it was discoveredthat when this N-terminal amino acid or analog thereof is characterizedby a side chain bearing an ionizable acidic group at physiological pH,an entirely new class of zinc metallopeptidase inhibitors, morespecifically zinc metallopeptidase inhibitors selective as PHEXinhibitors, can be delineated.

The present invention relates to derivatives of succinic and glutaricacids and analogs thereof useful as inhibitors of PHEX, as well as tomethods of administering them.

More specifically, the present invention relates to succinic andglutaric acids and derivatives or analogs thereof, characterized by thepresence of an ionizable acidic group (D) at physiological pH and by thepresence of a zinc ligand or zinc ligand bearing moiety (A) linked via alinker (B) to the acid residue, its derivative or analog thereof. Thecompounds of the present invention can be illustrated by the generalstructure shown below in Formula I:

wherein:

A is a zinc ligand or zinc ligand bearing moiety selected from the groupconsisting of:

B is

or absent;

R is hydrogen or lower alkyl;

R₁ is hydrogen or lower alkyl;

R₂ is hydrogen, or lower alkyl;

R₁, when v=1, may be connected to the carbon bearing R₂ to form analkylene bridge of 1 carbon atom, representing with the carbon atom towhich it is attached a cyclopropane ring;

R₂, when v=1, may be connected to the carbon bearing R₁ to form analkylene bridge of 1 carbon atom representing with the carbon atom towhich it is attached a cyclopropane ring;

R3 is hydrogen or lower alkyl;

R1, when v=1, may be connected to the carbon bearing R3 to form analkylene bridge of 1 carbon atom, representing with the carbon atom towhich it is attached a cyclobutane ring;

R3, when v=1, may be connected to the carbon bearing R1 to form analkylene bridge of 1 carbon atom, representing with the carbon atom towhich it is attached a cyclobutane ring;

R₁ and R₃, when v=1, may be connected together to form an alkylenebridge of 2 carbon atoms representing with the carbon atoms to whichthey are attached a cyclopentane ring;

R₁ and R₃, when v=0, may be connected together to form an alkylenebridge of 3 carbon atoms representing with the carbon atoms to whichthey are attached a cyclopentane ring;

R₁ and R₃, when v=0, may be connected together to form an alkylenebridge of 4 carbon atoms representing with the carbon atoms to whichthey are attached a cyclohexane ring;

R₁ and R₃, when v=1, may be connected together to form an alkylenebridge of 3 carbon atoms representing with the carbon atoms to whichthey are attached a cyclohexane ring;

R₄ is lower alkyl, substituted lower alkyl, cycloalkyl-(CH₂)_(w)—,aryl-(CH₂)_(w)—, substituted aryl-CH₂)_(w)— or heteroaryl-(CH₂)_(w)—;

R and R₄ may be connected together to form an alkylene bridge of 3carbon atoms representing with the nitrogen and carbon atoms to whichthey are attached a pyrrolidine ring;

R and R₄ may be connected together to form an alkylene bridge of 4carbon atoms representing with the nitrogen and carbon atoms to whichthey are attached a piperidine ring;

R₅ is hydrogen, lower alkyl, substituted lower alkyl,cycloalkyl-(CH₂)_(x)—, aryl-(CH₂)_(x)—, substituted aryl-(CH₂)_(x)—, orheteroaryl-(CH₂)_(x)—;

R₆ is hydrogen, R₇—CO—, or R₁₂—S—;

R₇ is alkyl, substituted alkyl, cycloalkyl-(CH₂)_(y), aryl-(CH₂)_(y)—,substituted aryl-(CH₂)_(y)— or heteroaryl-(CH₂)_(y)—;

R₈ and R₉ are independently selected from the group consisting ofhydrogen, alkyl, substituted alkyl, aryl-(CH₂)_(y)—, substitutedaryl-(CH₂)_(y)—, heteroaryl-(CH₂)_(y)—,

R₁₀ is alkyl, substituted alkyl, cycloalkyl-(CH₂)_(y), aryl-(CH₂)_(y)—,substituted aryl-(CH₂)_(y)— or heteroaryl-(CH₂)_(y)—;

R₁₁ is hydrogen or lower alkyl;

the carbon bearing R₁ and the nitrogen bearing R₁₁, when v=1, may bedirectly connected together to form an azetidine ring;

R₁ and R₁₁, when v=0, may be connected together to form an alkylenebridge of 3 carbon atoms representing with the nitrogen and carbon atomsto which they are attached a piperidine ring;

R₁ and R₁₁, when v=1, may be connected together to form an alkylenebridge of 2 carbon atoms representing with the nitrogen and carbon atomsto which they are attached a piperidine ring;

R₂ and R₁₁, when v=1, may be connected together to form an alkylenebridge of 2 carbon atoms representing with the nitrogen and carbon atomsto which they are attached a pyrrolidine ring; the alkylene bridge maybe substituted by a lower alkyl or alkenyl group at either carbon;

R₁₂ is alkyl, substituted alkyl, cycloalkyl-(CH₂)_(y)—, aryl-(CH₂)_(y)—,substituted aryl-(CH₂)_(y)—, heteroaryl-(CH₂)_(y)—,

in which case —S—R₁₂ completes a symmetrical disulfide;

R₁₃ is hydrogen, lower alkyl, cycloalkyl or phenyl;

R₁₄ is hydrogen, lower alkyl, lower alkoxy or phenyl;

R₁₅ is lower alkyl or aryl-(CH₂)_(y)—;

D is —COOH, —SO₂H, —SO₃H, —PO₃H₂; —OSO₃H or —OPO₃H₂;

E is hydrogen, R₁₂, —COOH, —CONH₂, —CONH(lower alkyl), —CON(loweralkyl)₂, —CONH—(CH₂)_(z)-aryl, —CON(—(CH₂)_(z)-aryl)₂, —CO-amino acid,—CH₂COOH, CH₂OH, —CH₂CH₂OH, or —COOR₁₆;

R₁₆ is as previously defined for R₈ and R₉;

C is carbon;

H is hydrogen;

O is oxygen;

N is nitrogen;

S is sulfur;

P is phosphorus;

v is zero or one;

w is zero or an integer ranging from 1 to 4;

x is an integer ranging from 1 to 4;

y is zero or an integer ranging from 1 to 6; and

z is zero, one, two or three.

The present invention also relates to the use of PHEX inhibitors for theidentification of PHEX substrates and to methods of identifying PHEXsubstrates.

In particular, it relates to a method for identifying PHEX substratescomprising contacting a candidate compound with PHEX in the presence andin the absence of a PHEX inhibitor of the present invention; assessingPHEX biological activity on the candidate compound in the presence andin the absence of the PHEX inhibitor, wherein the candidate compound isselected as a PHEX substrate when PHEX biological activity is measurablyhigher in the absence versus in the presence of the PHEX inhibitor. Italso relates to a use of a PHEX inhibitor of the present invention, foridentifying PHEX substrates comprising contacting a candidate with PHEXin the presence and in the absence of the PHEX inhibitor; and assessingPHEX biological activity on the candidate in the presence and in theabsence of the PHEX inhibitor, wherein the candidate compound isselected as a PHEX substrate when PHEX biological activity is measurablyhigher in the absence versus in the presence of the compound.

It also relates to a method for stimulating bone mass formation in amammal comprising inhibiting PHEX with an effective amount of a PHEXinhibitor of the present invention. It also relates to the use of a PHEXinhibitor of the present invention for stimulating bone mass formationin a mammal.

It also relates to a method for treating or preventing a disease orcondition associated with a phosphate metabolism defect comprisingadministering an effective amount of PHEX inhibitor of the presentinvention to a mammal in need thereof. It also relates to uses a PHEXinhibitor of the present invention for treating or preventing a diseaseor condition associated with a phosphate metabolism. In specificembodiments, the disease or condition is selected from the groupconsisting of hyperphosphatemia, hyperparathyroidism and renalinsufficiencies.

It also relates to a method for inhibiting PHEX comprising contactingPHEX with an inhibitory amount of a PHEX inhibitor of the presentinvention. It also relates to a use of a PHEX inhibitor of the presentinvention for inhibiting PHEX.

In addition, the present invention relates to methods of administrationof the PHEX inhibitors as described herein.

Moreover, the present invention relates to the use of pharmaceuticalpreparations comprising an effective amount of a PHEX inhibitor inmedical conditions such as hyperphosphatemia, hyperparathyroidism, inconditions related to renal insufficiencies such as renal osteodystrophyas well as in conditions wherein the metabolism of phosphate needs to bemanaged. In addition, the present invention relates to the use ofinhibitors of PHEX capable of providing for faster bone-massregeneration following bone-fractures, the implantation of orthopedicprostheses and the implantation of dental prostheses or after loss ofbone mass as a result of bone diseases such as osteoporosis.

The present invention also relates to pharmaceutical preparationcomprising an effective amount of a PHEX inhibitor in an admixture witha physiologically-acceptable carrier or excipient.

The terms “inhibiting,” “reducing” or “prevention,” or any variation ofthese terms, when used in the claims and/or the specification includesany measurable decrease or complete inhibition to achieve a desiredresult.

The use of the word “a” or “an” when used in conjunction with the term“comprising” in the claims and/or the specification may mean “one,” butit is also consistent with the meaning of “one or more,” “at least one,”and “one or more than one.”

It is contemplated that any embodiment discussed in this specificationcan be implemented with respect to any method or composition of theinvention, and vice versa. Furthermore, compositions and kits of theinvention can be used to achieve methods of the invention.

Throughout this application, the term “about” is used to indicate that avalue includes the standard deviation of error for the device or methodbeing employed to determine the value.

The use of the term “or” in the claims is used to mean “and/or” unlessexplicitly indicated to refer to alternatives only or the alternativesare mutually exclusive, although the disclosure supports a definitionthat refers to only alternatives and “and/or.”

As used in this specification and claim(s), the words “comprising” (andany form of comprising, such as “comprise” and “comprises”), “having”(and any form of having, such as “have” and “has”), “including” (and anyform of including, such as “includes” and “include”) or “containing”(and any form of containing, such as “contains” and “contain”) areinclusive or open-ended and do not exclude additional, unrecitedelements or method steps.

Further scope and applicability will become apparent from the detaileddescription given hereinafter. It should be understood however, thatthis detailed description, while indicating preferred embodiments of theinvention, is given by way of illustration only, since various changesand modifications within the spirit and scope of the invention willbecome apparent to those skilled in the art.

BRIEF DESCRIPTION OF THE DRAWINGS

Having thus generally described the invention, reference will now bemade to the accompanying figures, showing by way of illustration apreferred embodiment thereof, and in which:

FIG. 1 shows the chemical structure of the PHEX inhibitor MH-2-64C(compound of Example 126);

FIG. 2 illustrates in vitro protection of PHEX substrate PTHrP(107-132)by inhibitor MH-2-64C (compound of Example 126);

FIG. 3 illustrates the effect of adding decreasing concentrations of thePHEX inhibitor MH-2-64C (compound of Example 126) to a culture medium ofprimary rat calvaria osteoblasts, on mineralization;

FIG. 4 represents scanning electron microscopy pictures of lesions inrat mandibles repaired in the presence or the absence of the PHEXinhibitor MH-2-64C (compound of Example 126), as well as illustratingquantification of bone fraction; and

FIG. 5 illustrates a 3D-reconstruction by micro computed tomography oflesions in the rat mandibles, repaired in the presence or the absence ofthe PHEX inhibitor MH-2-64C (compound of Example 126) presented in FIG.4.

Other objects, advantages and features of the present invention willbecome more apparent upon reading of the following non-restrictivedescription of preferred embodiments with reference to the accompanyingdrawings, which is exemplary and should not be interpreted as limitingthe scope of the present invention.

DETAILED DESCRIPTION OF THE INVENTION

As used herein, “sPHEX” refers to a secreted soluble form of human PHEXwhich causes the hydrolysis of PTHrP₁₀₇₋₁₃₉ by cleaving peptide bonds onthe amino-terminal side of aspartate amino acid residues. The presentinvention is to be understood as comprising the inhibition of allenzymes having such activity, a non-limiting example of which ismembrane-bound PHEX.

As used herein, the term “PHEX inhibitor” comprises any compound thatinhibits the enzymatic action of sPHEX.

The term “derivative” as used herein, is understood as being a substancewhich comprises the same basic carbon skeleton and carbon functionalityin its structure as a given compound, but can also bear one or moresubstituents or rings. Non-limiting examples of derivatives of succinicacid include: 2-amino succinic acid (aspartic acid), dimethyl succinate,2-benzyl succinic acid, succinic anhydride,1-aminocyclopentane-1,2-dicarboxylic acid and 2,3-piperidinedicarboxylicacid.

As used herein, the terminology PHEX “biological activity” is meant toinclude enzymatic activity and binding of PHEX to other moleculesincluding inhibitors and substrates.

The term “analog” as used herein, is understood as being a substancewhich does not comprise the same basic carbon skeleton and carbonfunctionality in its structure as a “given compound”, but which canmimic the given compound by incorporating one or more appropriatesubstitutions such as for example substituting carbon for heteroatoms.Non-limiting examples of “analogs” include: cysteic acid is an analog ofaspartic acid, O-phosphoserine, serine-O-sulfate and2-amino-4-phosphobutanoic acid are analogs of glutamic acid.

The term “alkyl” as used herein, is understood as being straight orbranched chain radicals having up to seven carbon atoms. The term “loweralkyl” as used herein, is understood as being straight or branchedradicals having up to four carbon atoms and is a preferred sub-groupingfor the term “alkyl”.

The term “substituted alkyl” as used herein, is understood as being suchstraight or branched chain radicals having up to 7 carbon atoms whereinone or more, preferably one, two, or three hydrogen atoms have beenreplaced by a substituent selected from the group consisting of hydroxy,amino, cyano, halogen, trifluoromethyl, —NH(lower alkyl), —N(loweralkyl)₂, lower alkoxy, lower alkylthio, and carboxy, aryl andheteroaryl.

The terms “lower alkoxy” and “lower alkylthio” as used herein, areunderstood as being such lower alkyl groups as defined above attached toan oxygen or sulfur atom.

The term “cycloalkyl” as used herein, is understood as being saturatedrings of 3 to 7 carbon atoms.

The term “alkenyl” as used herein, is understood as being straight orbranched chain radicals of 3 to 7 carbon atoms having one or two doublebonds. Preferred “alkenyl” groups are straight chain radicals of 3 to 5carbon atoms and having one double bond.

The term “substituted alkenyl” as used herein, is understood as beingsuch straight or branched radicals of 3 to 7 carbon atoms having one ortwo double bonds and wherein a hydrogen atom has been replaced by asubstituent selected from the group consisting of hydroxy, amino, halo,trifluoromethyl, cyano, —NH(lower alkyl), —N(lower alkyl)₂, loweralkoxy, lower alkylthio, and carboxy.

The term “alkylene” as used herein, is understood as being divalentstraight or branched chain radicals having up to seven carbon atoms(i.e. —CH₂—, —(CH₂)₂—, —(CH₂)₃—, —(CH₂)₄—, —CH₂—CH(CH₃)—, etc.).

The term “aryl” as used herein, is understood as being phenyl,1-naphthyl, and 2-naphthyl. The term “substituted aryl” as used herein,is understood as being phenyl, 1-naphthyl and 2-naphthyl having asubstituent selected from the group consisting of phenyl, heteroaryl,lower alkyl, lower alkoxy, lower alkylthio, halo, hydroxy,trifluoromethyl, amino, —NH(lower alkyl), and —N(lower alkyl)₂, as wellas being mono-, di- and tri-substituted phenyl, 1-naphthyl, and2-naphthyl comprising substituents selected from the group consisting ofmethyl, methoxy, methylthio, halo, hydroxy, and amino.

The term triphenylmethyl is herein abbreviated as Trt (trityl).

The term “heteroaryl” as used herein, is understood as being unsaturatedrings of five or six atoms containing one or two O- and/or S-atomsand/or one to four N-atoms, provided that the total number ofhetero-atoms in the ring is 4 or less. The heteroaryl ring is attachedby way of an available carbon or nitrogen atom. Preferred heteroarylgroups include 2-, 3-, or 4-pyridyl, 4-imidazolyl, 4-thiazolyl, 2- and3-thienyl, and 2- and 3-furyl. The term “heteroaryl” as used herein, isunderstood as also including bicyclic rings wherein the five or sixmembered ring containing O, S and N-atoms as defined above is fused to abenzene or pyridyl ring. Preferred bicyclic rings include but are notlimited to 2- and 3-indolyl as well as 4- and 5-quinolinyl. The mono orbicyclic heteroaryl ring can also be additionally substituted at anavailable carbon atom by a substituent selected from the groupconsisting of lower alkyl, halo, hydroxy, benzyl and cyclohexylmethyl.Additionally, if the mono or bicyclic ring has an available N-atom, thensuch an atom can also be substituted by one of the N-protecting groupssuch as N-carbamates, N-phenylsulfenyl, N-phenylsulfonyl,N-2,4-dinitrophenyl, N-lower alkyl, N-benzyl, or N-benzhydryl or anyother applicable group known in the art (T. W. Greene, P. G. M. Wuts:Protective Groups in Organic Synthesis, 2^(nd) Edition, John Wiley &Sons, NY, 1991).

The terms “halogen” or “halo” as used herein, is understood as beingchlorine, bromine, fluorine and iodine.

The term “salt(s)” as used herein, is understood as being acidic and/orbasic salts formed with inorganic and/or organic acids and bases.Zwitterions (internal or inner salts) are understood as being includedwithin the term “salt(s)” as used herein, as are quaternary ammoniumsalts such as alkylammonium salts. Nontoxic, pharmaceutically acceptablesalts are preferred, although other salts may be useful, as for examplein isolation or purification steps.

Examples of acid addition salts include but are not limited to acetate,adipate, alginate, aspartate, benzoate, benzenesulfonate, bisulfate,butyrate, citrate, camphorate, camphorsulfonate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, fumarate, glucoheptanoate,glycerophosphate, hemisulfate, heptanoate, hexanoate, hydrochloride,hydrobromide, hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, oxalate,pectinate, persulfate, 3-phenylpropionate, picrate, pivalate,propionate, succinate, tartrate, thiocyanate, tosylate, and undecanoate.

Examples of basic salts include but are not limited to ammonium salts;alkali metal salts such as sodium, lithium, and potassium salts;alkaline earth metal salts such as calcium and magnesium salts; saltscomprising organic bases such as amines (e.g., dicyclohexylamine,alkylamines such as t-butylamine and t-amylamine, substitutedalkylamines, aryl-alkylamines such as benzylamine, dialkylamines,substituted dialkylamines such as N-methyl glucamine (especiallyN-methyl D-glucamine), trialkylamines, and substituted trialkylamines);and salts comprising amino acids such as arginine, lysine and so forth.The basic nitrogen-containing groups may be quaternized with agents suchas lower alkyl halides (e.g. methyl, ethyl. propyl, and butyl chlorides,bromides and iodides), dialkyl sulfates (e.g. dimethyl, diethyl,dibutyl, and diamyl sulfates), long chain halides (e.g. decyl, lauryl,myrtistyl and stearyl chlorides, bromides and iodides), arylalkylhalides (e.g. benzyl and phenethyl bromides), and others known in theart.

Prodrugs and solvates of the PHEX inhibitors of the present inventionare also contemplated herein. The term “prodrug” as used herein, isunderstood as being a compound which, upon administration to a subject,undergoes chemical conversion by metabolic or chemical processes toyield a compound of the Formula I, or a salt and/or solvate thereof(Bundgaard, 1991; Bundgaard, 1985). Solvates of the compounds of FormulaI are preferably hydrates.

All possible stereoisomers of the PHEX inhibitors of the presentinvention are contemplated as being within the scope of the presentinvention. Individual stereoisomers of the compounds of the presentinvention may, for example, be substantially free of otherstereoisomers, or may be admixed, for example, as racemates or admixedwith other selected or all other stereoisomers. The chiral centers ofthe PHEX inhibitors of the present invention can have the S- or theR-configuration, as defined by the IUPAC 1974 Recommendations.

The compounds of Formula I wherein A, B, D, E, R, R₁, R₂, R₃, R₄ and vare as defined above, can be prepared by reacting a compound of FormulaII,

wherein R, R₄ and E are as defined above, and wherein E and R₄ (ifneeded) are suitably protected by a conventional protecting group so asnot to interfere with the coupling reaction using conventional peptidesynthesis methodology, with a derivative of Formula III

-   -   wherein A, B, D, R₁, R₂, R₃ and v are as defined above, and        wherein A and D are suitably protected so as not to interfere        with the coupling reaction, using conventional peptide synthesis        methodology.

The compound of Formula III wherein B is NR₁₁ and wherein A, D, R₁, R₂,R₃ and v are as defined above, can be prepared via a condensationreaction between a compound of Formula IV,

suitably protected at the carboxyl and D groups and wherein R₁, R₂, R₃,R₁₁, D and v are as defined above, and a suitably protected precursor ofA to give a compound of Formula IIIa, suitably protected at the carboxyland D groups, wherein A, D, R₁, R₂, R₃, R₁₁, and v are as describedabove.

When the compound of Formula IIIa includes a group A which isR₆S—CHR₅—C(═O)— wherein R₅ and R₆ are as defined above, it can beprepared by peptidic condensation of carboxy- and D-protected compoundIV with an S-protected (and if needed, R₅ and R₆ protected) thioglycolicacid of Formula:

When the compound of Formula IIIa includes a group A which isR₆S—CHR₅—(CH₂)_(w)— wherein R₅ and R₆ are as defined above and whereinw=1, it can be prepared by reaction of carboxy- and D-protected compoundIV with a thiiran of Formula (protected at R₅ if needed).

When the compound of Formula IIIa includes a group A which isTrtO—NH—C(═O)— wherein Trt is a trityl protecting group, it can beprepared by reaction of carboxy- and D-protected compound IV withtrichloromethyl chloroformate followed by trapping of the resultingchloroformamide with O-tritylhydroxylamine.

When the compound of Formula IIIa includes a group A which isR₈O—C(═O)—CHR₅— wherein R₅ and R₈ are as defined above, it can beprepared by reductive amination involving carboxy- and D-protectedcompound IV and an α-keto ester (or the corresponding acid) protected atR₈ and/or R₅ (if needed) of Formula:

Alternatively, the above-mentioned compound IIIa can be prepared byreaction with a triflate (protected, if needed, at R₈ and R₅) offormula:

When the compound of Formula IIIa includes a group A which isTrt-O—NH—C(═O)CHR₅— wherein Trt is a trityl protecting group and R₅ isas defined above, it can be prepared by reaction of compound IIIIa,protected at the carboxyl and D groups, wherein A is R₈—O—C(═O)—CHR₅—and wherein R₅ (protected if needed) and R₈ are as defined above, withO-trityl-protected hydroxylamine.

When the compound of Formula IIIa includes a group A which isR₉O—P(═O)R₁₀— wherein R₉ and R₁₀ are as defined above, it can beprepared by condensation of carboxy- and D-protected compound IV with aphosphonochloridate (with R₉ and R₁₀ protected, if needed) of theFormula:

When the compound of Formula IIIa includes a group A which is(R₉O)₂P(═O)—CHR₅— wherein R₅ and R₉ are as defined above, it can beprepared by reacting compound IV or a salt thereof with an aldehyde offormula R₅—CHO having R₅ protected if needed, followed by treating theproduct of this reaction with a phosphite of Formula:

When the compound of Formula IIIa includes a group A which isR₁₀R₉OP(═O)—CHR₅—C(═O)— wherein R₅, R₉ and R₁₀ are as defined above, itcan be prepared by reacting carboxy- and D-protected compound IV with aphosphinyl acetic acid derivative of Formula:

(with R₅, R₉, and R₁₀ protected if needed) following known amide bondforming procedures.

In the preceding set of reactions, a reaction sequence was followed inwhich compounds of Formula III were assembled by formation of the A-Bbond, followed by coupling of the compounds of Formula III to compoundsof Formula II to provide compounds of Formula I. The reverse sequence inwhich the amide bond to compounds of Formula II is first formed,followed by the formation of the A-B bond, is equally feasible.

The compounds of Formula II wherein E, R and R₄ are defined as above,can in many cases be obtained from commercial sources, particularly whenit is a natural or non-natural α-amino acid or its decarboxylationproduct or when E is hydrogen or —COOH. In the case where E is a simpleprimary, secondary or tertiary amide, a peptide bond to an amino acid,an ester or a hydroxymethyl group, these compounds can all be preparedfrom the compound wherein E is —COOH by procedures well known in theart. Finally, the carbon chain elongated analogs of compound II whereinE is —CH₂COOH or —CH₂CH₂OH can also be prepared from compounds wherein Eis —COOH by procedures well known in the art.

Many compounds of Formula IV which are substituted derivatives andanalogs of α-aminosuccinic acid (aspartic acid) and α-amino glutaricacid (glutamic acid) are commercially available and can be found in TheProtein Synthesis Database (PSD) from The Biotechnology ResearchInstitute (BRI) of The National Research Council of Canada (NRCC).Specific examples of compounds of Formula IV, included herein in theirunprotected and variously protected forms are: D-Aspartic acid,L-Aspartic acid, DL-Aspartic acid, N-Methyl-D-aspartic acid,N-Methyl-L-aspartic acid, alpha-Methyl-D-aspartic acid,DL-threo-beta-Methylaspartic acid, D-(+)-threo-beta-Hydroxyasparticacid, DL-threo-beta-Hydroxyaspartic acid,L-(+)-threo-beta-Hydroxyaspartic acid, L-Cysteinesulfinic acid,L-Cysteic acid, L-(+)-2-Amino-3-phosphonopropionic acid,D-(−)-2-Amino-3-phosphonopropionic acid, Cis-2,3-Piperidinedicarboxylicacid, (±)-1-Aminocyclopentane-cis-1,2-dicarboxylic acid,(±)-1-Aminocyclopentane-trans-1,2-dicarboxylic acid,(±)-1-Aminocyclohexane-cis-1,2-dicarboxylic acid,(±)-1-Aminocyclohexane-trans-1,2-dicarboxylic acid, L-Glutamic acid,D-Glutamic acid, DL-Glutamic acid, (2R,4R)-(+)-Azetidine-2,4-dicarboxylic acid, (2S,4S)-(+)-Azetidine-2,4-dicarboxylic acid, γ-Methylene-DL-glutamic acid,1-Aminocyclobutane-cis-1,3-dicarboxylic acid,1-Aminocyclobutane-trans-1,3-dicarboxylic acid, (2S, 1′R,2′S)-2-(Carboxycyclopropyl)glycine, (2S, 1′S,2′R)-2-(Carboxycyclopropyl)glycine, (2S, 1′S,2′S)-2-(Carboxycyclopropyl)glycine, N-Methyl-L-Glutamic acid,α-Methyl-DL-Glutamic acid, (2S, 4R)-4-Methylglutamic acid,4-Fluoroglutamic acid, cis-2,4-Piperidinedicarboxylic acid, (1R,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid, (1R,3S)-1-Aminocyclopentane-1,3-dicarboxylic acid, (1S,3R)-1-Aminocyclopentane-1,3-dicarboxylic acid, (1S,3S)-1-Aminocyclopentane-1,3-dicarboxylic acid, (2S)-α-Ethylglutamicacid, D-Homocysteic acid, L-Homocysteic acid,L(+)-2-Amino-4-phosphonobutanoic acid, D(+)-2-Amino-4-phosphonobutanoicacid, L-Serine O-sulfate, O-Phospho-L-serine, O-Phospho-D-serine,(±)-1-Aminocyclohexane-cis-1,3-dicarboxylic acid, L-γ-carboxyglutamicacid, D-γ-carboxyglutamic acid, (S)-2-Amino-2-methyl-4-phosphonobutanoicacid, O-Phospho-L-threonine, O-Phospho-DL-threonine, Kainic acid, andDihydrokainic acid.

The compound of Formula III wherein B is CH₂, A is R₆S—CHR₅—(CH₂)_(w)—,D, R₅ and R₆ are as defined above, wherein R₁, R₂ and R₃ are hydrogen,w=0 and v=0 or 1, and which is hereinafter specified as compound ofFormula IIIb, can be prepared according to the reaction sequenceillustrated in Scheme 1 (appropriate protecting groups are used asneeded). The sequence begins with succinic acid monomethyl ester, thedianion of which is alkylated with electrophile V or VI to give compoundof the formula VII. The latter is reduced to the corresponding aldehydeVIII using 9-BBN. Grignard reaction on aldehyde VIII gives compound IXwhich is converted to the corresponding thioester X under Mitsunobuconditions. Finally, compound IIIb wherein B is CH₂, A isR₆—S—CHR₅—(CH₂)_(w)—, D, R₅ and R₆ are as defined above, wherein R₁, R₂and R₃ are hydrogen and wherein w=0 and v=0 or 1, is obtained by basichydrolysis of thioester X followed by the introduction of R₆ by standardmethods.

The compound of Formula III wherein B is CH₂, A is R₆S—CHR₅—(CH₂)_(w)—,D, R₅ and R₆ are as defined above, wherein R₁, R₂ and R₃ are hydrogenand wherein w=1 and v=0 or 1, and which is hereinafter referred to ascompound of Formula IIIc, can be prepared according to the reactionsequence illustrated in Scheme 2. Scheme 2 involves the same reactionsequence as the one previously illustrated in Scheme 1, except that itstarts from the next higher carbon homologue, i.e., glutaric acidmonomethyl ester.

The compound of Formula III wherein B is CH₂, A is TrtO—NH—C(═O)—, D isas defined above, wherein R₁, R₂ and R₃ are hydrogen and wherein v=0 or1 and which is hereinafter specified as compound of Formula IIId, can beprepared by reacting compound VIIa or VIIa-1 with O-tritylhydroxylamineunder peptide bond forming conditions, followed by base hydrolysis ofthe methyl ester.

The compound of Formula III wherein B is CH₂, A is R₈—O—C(═O)—CHR₅—, D,R₅ and R₈ are as defined above, wherein R₁, R₂ and R₃ are hydrogen andwherein v=0 or 1, and which is hereinafter referred to as compound ofFormula IIIe, can be prepared by the reaction sequence illustrated inScheme 3, starting from aldehyde VIIb or VIIIb-1 and where R₅ and R₈ areprotected if needed.

The sequence begins with the alkylation of aldehyde VIIIb or VIIIb-1with R₅—Br to give compound XI. Pyridinium dichromate (PDC) oxidation ofthe aldehyde gives the corresponding acid XII. The latter is thenconverted to its tert-butyl ester using tert-butyl2,2,2-trichloroacetimidate, followed by the selective hydrolysis of themethyl ester using aqueous base to give compound IIIe.

The compound of Formula III wherein B is CH₂, A is TrtO—NH—C(═O)—CHR₅—,D and R₅ are as defined above, wherein R₁, R₂ and R₃ are hydrogen andwherein v=0 or 1 and which is hereinafter referred to as compound ofFormula IIIf, can be prepared by reacting compound XIIb or XIIb-1(Scheme 3) (having R₅ protected if needed) with O-tritylhydroxylamineunder peptide bond forming conditions, followed by base hydrolysis ofthe methyl ester.

The compound of Formula III wherein B is CH₂, A is R₁₀R₉OP(═O)—, whereinD, R₉ and R₁₀ are as defined above, wherein R₁, R₂ and R₃ are hydrogenand wherein v=0 or 1, and which is hereinafter specified as compound ofFormula IIIg, can be prepared according to known procedures (U.S. Pat.No. 4,168,267).

The compound of Formula III wherein B is CH₂, A is (R₉O—)₂P(═O)—CHR₅—,wherein D, R₅ and R₉ are as defined above, wherein R₁, R₂ and R₃ arehydrogen and wherein v=0 or 1 and which is hereinafter specified ascompound of Formula IIIh, can be prepared according to Scheme 4 where R₅and R₉ can be protected if needed.

The synthetic sequence involves the conversion of alcohol IXa or Ixa-1(Scheme 1) to its corresponding bromide using CBr₄/PPh₃ followed by anArbuzov reaction and basic hydrolysis of the methyl ester.

The compound of Formula III wherein B is absent, A isR₆—S—CHR₅—(CH₂)_(w)—, wherein D, R₅ and R₆— are as defined above,wherein R₁, R₂ and R₃ are hydrogen and wherein w=0 and v=0 or 1, andwhich is hereinafter specified as compound of Formula IIIi, can beprepared according to the reaction sequence illustrated in Scheme 5starting from diethyl malonate and where R₅ and R₆ can be protected ifneeded.

This sequence involves the same reactions as those previously describedin Schemes 1 and 2 to prepare the higher homologues IIIb and IIIc.

The compound of Formula III wherein B is absent, A is TrtONH—C(═O)—, Dis as defined above, wherein R₁, R₂ and R₃ are hydrogen and wherein v=0or 1, and which is hereinafter referred to as compound of Formula IIIj,can be prepared by reacting compound VIIc or VIIc-1 (Scheme 5) withO-trityl hydroxylamine under peptide bond forming conditions followed bybase hydrolysis of the ethyl ester.

The compound of Formula III wherein B is absent, A is R₈—O—C(═O)—CHR₅—,wherein D, R₅ and R₈ are as defined above, wherein R₁, R₂ and R₃ arehydrogen and wherein v=0 or 1, and which is hereinafter specified ascompound of the Formula IIIk, can be prepared according to the reactionsequence illustrated in Scheme 6, where R₅ and R₈ can be protected ifneeded.

Thus, opening of maleic anhydride with the sodium alkoxide R₈ONa givesthe half ester which upon treatment with trityl chloride affords themixed maleic diester. Conjugate addition/enolate trapping givespredominantly the correct succinate regioisomer as predicted based onsteric control grounds. Finally, selective hydrolysis of the tritylester affords the target compound IIIk.

The compound of Formula III wherein B is absent, A isBnO—NH—C(═O)—CHR₅—, wherein D and R₅ are as defined above, wherein R₁,R₂ and R₃ are hydrogen and wherein v=0 or 1, and which is hereinafterreferred to as compound of Formula IIII, can be prepared by reactingcompound XII, obtained by selective hydrolysis of XI (Scheme 6), withO-benzylhydroxylamine under peptide bond forming conditions followed bycleavage of the trityl ester.

The compound of Formula III wherein B is absent, A is R₁₀R₉O—P(═O)—,wherein D, R₉ and R₁₀ are as defined above, wherein R₁, R₂ and R₃ arehydrogen and wherein v=0 or 1, and which is hereinafter specified ascompound of Formula IIIm, can be prepared according to known procedures(U.S. Pat. No. 4,168,267).

The compound of Formula III wherein B is absent, A is(R₉—O—)₂P(═O)—CHR₅—, wherein D, R₅ and R₉ are as defined above, whereinR₁, R₂ and R₃ are hydrogen and wherein v=0 or 1, and which ishereinafter specified as compound of Formula IIIn, can be preparedaccording to Scheme 7 where R₅ can be protected if needed.

The synthetic sequence in scheme 7 involves the alkylation ofcommercially available tert-butyl diethylphosphonoacetate to give themixed ester XIII. Condensation of the in-situ generated anion of XIIIwith aldehyde R₅CHO affords the substituted acrylate derivative XIV.Arbuzov-type conjugate addition to acrylate XIV followed by TFA removalof the tert-butyl ester group furnishes target compound IIIn.

The compound of Formula III wherein B is CH₂, A is R₁₀R₉OP(═O)—CHR₅—,wherein D, R₅, R₉ and R₁₀ are as defined above, wherein R₁, R₂ and R₃are hydrogen and wherein v=0 or 1, and which is hereinafter specified ascompound of Formula IIIo, can be prepared according to Scheme 8 whereR₅, R₉ and R₁₀ can be protected if needed.

The synthetic sequence in Scheme 8 involves first halogenation of thestarting alcohol IXa or IXa-1 to give the corresponding bromoester XV.The later is converted to the monosubstituted phosphinic acid which isthen protected as its ester XVI by reacting with R₉OH. Finally, Arbuzovreaction with alkyl bromide R₁₀Br on diester XVI followed by hydrolysisof the methyl ester gives the target compound IIIo.

The compound of Formula III wherein B is CH₂, A is R₁₀R₉OP(═O)—CHR₅—CH₂,wherein D, R₅, R₉ and R₁₀ are as defined above, wherein R₁, R₂ and R₃are hydrogen and wherein v=0 or 1, and which is hereinafter specified ascompound of Formula IIIp, can be prepared according to Scheme 9, whereR₅, R₉ and R₁₀ can be protected if needed. Scheme 9 involves the samereaction sequence as the one previously illustrated in Scheme 8, exceptthat it starts from alcohol IXb or IXb-1 instead of IXa or IXa-1.

The compound of Formula V wherein D is —COOR₅ can in some cases beobtained from commercial sources or simply prepared by reacting thecorresponding alcohol R₅OH with commercially available bromoacetylbromide or chloride.

The compound of Formula V wherein D is —P(═O)(OR₅)₂ can in some cases beobtained from commercial sources or simply prepared by reacting thecorresponding alcohol R₅OH with commercially availablechloromethylphosphonic dichloride followed by exchanging the chloride ofthe chloromethyl group for bromide using sodium bromide.

In those cases where D is SO₃H, its introduction into the compounds ofFormula I and III, as well as in their precursors, can be achieved by asequence of reactions that does not involve the use of a compound ofFormula V to reach intermediate (VIIa), v=0. The intermediate of Formula(VIIa), v=0, can be prepared according to the reaction sequenceillustrated in Scheme 10.

Scheme 10, which is based on known literature precedents, involves theuse of an α-methylenation reaction followed by conjugate addition of athiocarboxylic acid to the resulting acrylate ester (See for exampleRoques et al. U.S. Pat. No. 4,513,009; Ondetti et al. U.S. Pat. Nos.4,105,776 and 4,339,600; Haslanger et al. U.S. Pat. No. 4,801,609 andDelaney et al. U.S. Pat. No. 4,722,810). Methanolysis of the resultingthioester gives the corresponding thiol which is oxidized to itsdisulfide dimer. The latter is then oxidatively cleaved by chlorine togive the corresponding sulfonyl chloride compound which is converted toits neopentyl ester (VIIa), v=0 wherein D is —SO₃CH₂C(CH₃)₃, a protectedform of —SO₃H.

The compounds of formula (VI) (see for example Scheme 1) can be obtainedfrom commercial sources and can be protected using methods known in theart.

The compounds of the present invention may be prepared by standardtechniques known to those skilled in the art. Preferred procedures forthe synthesis of these compounds are described below.

Amide bonds in the following examples are generally prepared bycondensation between carboxylic acids and amines. Reagents capable ofeffecting this condensation include, but are not limited to, oxalylchloride, thionyl chloride, phosphoryl chloride, diarylphosphorylazides, diarylphosphoryl cyamide and carbodimides. Carbodiimides inconjunction with 1-hydroxybenzotriazole, N-hydroxysuccinimide, and otherreagents well known in the synthesis of amide bonds and peptides mayalso be utilized. Salts of the described carboxylic acids may generallybe used in place of the carboxylic acids. Likewise, salts of thedescribed amines may frequently be used in place of the amines.

In some examples, alkyl and arylalkyl esters have been utilized toprotect carboxylic acids, hydroxamic acids, phosphonic acids andphosphinic acids. The protecting groups can be removed bywell-established procedures. Thus t-butyl esters can be cleaved bymineral or organic acids such as HCl, HBr, or trifluoroacetic acid.Primary alkyl esters can be hydrolyzed with base. Benzyl esters can alsobe removed by hydrogenolysis (Greene, 1999).

Various peptide libraries are prepared by manual and/or automated liquidand solid phase synthesis techniques known in the art of combinatorialchemistry (Thompson et al.; 1996; Hermkens et al., 1996; Balkenhohl etal., 1996; Furka et al., 1996; Terrett et al., 1995). Instruments suchas the Advanced ChemTech 440 Multiple Organic Synthesizer (440 MOS) wereused to generate compound libraries in an automated fashion (Rivero etal., 1997). Among the various solid phase supports, the Wang and2-Cl-Trityl resins were most often used. Non-limiting examples of theside chain protecting groups used in the present invention include: Asp(^(t)Bu), Asn (Trt), Arg (Pmc), Glu (^(t)Bu), His (Trt), Ser (^(t)Bu),Thr (^(t)Bu), Trp (Boc), and Tyr (^(t)Bu).

General Procedures for the Solid Phase Peptide Synthesis:

In the solid-phase peptide synthesis techniques using Fmoc/^(t)Buchemistry (Atherton and Sheppard, 1987), the N-α-Fmoc group was cleavedwith 25% (v/v) of piperidine in DMF for 5 min with continuous mixingfollowed by another treatment with fresh reagent for 25 min withcontinuous mixing. The resin was then filtered and washed sequentiallywith DMF (3×), DCM (3×), MeOH (3×), DCM (3×), and finally with MeOH(3×). The coupling reactions were done twice using a 4-fold and then a2-fold molar excess of reagents in order to maximize efficiency. Afterwashing and drying, cleavage was done using different cocktail mixturesdepending on the type of resin and peptide sought. For example, cocktailA: TFA (94%): H₂O (2.5%): EDT (2.5%): TlS (1.0%) was added to the resinwith mixing for 2 h at room temperature. After filtration, the resin waswashed by a fresh aliquot of the cleavage cocktail.

The cleaved samples were obtained by evaporation on a speed-vac machineand the remaining residues were precipitated with a cold mixture ofether:hexane (1:2). The isolated peptides were washed with coldether:hexane (1:2, 2×), dissolved in H₂O and then lyophilized. In thosecases where some peptides gave fine crystals, centrifugation was used toisolate the products. Those samples which did not provide anyprecipitate were portioned between H₂O and the above organic mixture andthe aqueous layers were extracted (3×) with the above mixture and werethen lyophilized. In those cases where a particular protected peptidewith free carboxy terminus is needed for further elaboration, thecleavage was done using a mixture of CH₂Cl₂ (DCM) andhexafluoroisopropanol (HFIP), cocktail B, (DCM:HFIP, 4:1).

The library samples were checked for purity by HPLC and MS.

The names of the compounds described in the examples below weregenerated using the “Chemdraw Ultra™” (version 7.0.1) software ofCambridgeSoft.

EXAMPLE 1 (R)-2-Bromo-3-phenyl-propionic acid

Potassium bromide (24.49 g, 0.21 moles) was first dissolved in a 2.50 Naqueous solution of sulfuric acid (120 mL). To this stirring solutionwas then added D-phenylalanine (10.00 g, 60.54 mmoles). After theaddition, the mixture was cooled down to 0° C. and sodium nitrite (6.27g., 90.87 mmoles) was added in small portions during 30 minutes. Astrong bubbling was observed. The reaction was kept at this temperaturefor 50 minutes, and it was then allowed to proceed at room temperaturefor another 60 minutes. The crude product was isolated using standardwork up and the obtained residue was purified by flash chromatography(SiO₂, 30% ethyl acetate−70% hexanes) to give 7.20 g (51.9% yield) ofthe title compound as a yellowish oil: ¹H NMR (CDCl₃, 300 MHz) □ 10.86(br s, 1H), 7.31 (m, 5H), 4.48 (t, 1H), 3.51 (dd, 1H), 3.29 (dd, 1H);¹³C NMR (CDCl₃, 75 MHz) □175.29, 136.26, 129.10, 128.69, 127.42, 44.83,40.62.

EXAMPLE 2 (R)-2-Acetylsulfanyl-3-phenyl-propionic acid

The cesium thioacetate, prepared from 10.75 g of cesium carbonate and2.51 g of thioacetic acid, was taken into dry DMF (25 mL) and to thissolution was transferred the compound of example 1 (31.43 mmoles)previously dissolved in anhydrous DMF (25 mL). The mixture was shakenfor 20 hours at room temperature and the product was isolated usingstandard work up to give 6.68 grams (94.8% yield) of the crudethioacetate. The compound was directly used in the next step without anyfurther purification.

EXAMPLE 3 (S)-2-Mercapto-3-phenyl-propionic acid

To a stirring solution of the compound of example 2 (6.68 g, 29.78mmoles) in degassed methanol (350 mL) was added dropwise a degassed 10%aqueous solution of potassium carbonate (9.61 g, 69.56 mmoles). Themixture was shaken at room temperature for 3 hours and the product wasisolated using standard work up to give 5.26 g (96.9% yield) of a yellowoil which was used immediately in the next step without any furtherpurification.

EXAMPLE 4 (S)-3-Phenyl-2-tritylsulfanyl-propionic acid

To a stirring solution of the above thioacid (5.26 g, 28.86 mmoles) indry THF (20 mL) was transferred drop-wise a solution of triphenylmethylchloride (12.07 g, 43.30 mmoles) in dry THF (20 mL). The mixture wasstirred at room temperature for 24 hours and was then evaporated undervacuum. The crude residue was purified by flash chromatography (SiO₂,20% EtOAc:80% Hexanes) to afford 6.02 g (49.1% yield) of the desiredcompound as a white solid: ¹H NMR (CDCl₃, 300 MHz) □ 7.40 (m, 6H) 7.20(m, 12H), 6.88 (d, 2H), 3.14 (dd, 1H), 2.92 (dt, 1H), 2.48 (dd, 1H); ¹³CNMR (CDCl₃, 75 MHz) □ 178.93, 144.47, 137.81, 130.11, 129.58, 129.11,128.46, 127.42, 127.31, 68.98, 49.57, 39.54.

The following 2-tritylsulfanyl acids (compounds of examples 5–20) wereprepared following the same sequence as that described in examples 1–4,where the starting 2-bromo carboxylic acid is obtained by nitrous aciddeamination of the corresponding α-amino acid in the presence of HBr.

EXAMPLE 5 4-Phenyl-2-tritylsulfanyl-butyric acid

¹H NMR (CDCl₃, 400 MHz) δ 7.49 (m, 6H), 7.31–7.16 (m, 12H), 7.02 (m,2H), 2.99 (dd, 1H), 2.56–2.39 (m, 2H), 2.05–1.95 (m,1H), 1.66–1.59 (m,1H); ¹³C NMR (CDCl₃, 400 MHz) δ 178.6, 144.0, 140.4, 129.5, 128.2,127.8, 126.8, 125.9, 114.3, 95.7, 68.3, 46.8, 34.6, 33.4; MS(FAB-/LR/NBA) 437.0.

EXAMPLE 6 Phenyl-tritylsulfanyl-acetic acid

¹H NMR (CDCl₃, 400 MHz) δ 7.41–7.09 (m, 20H), 4.11 (s, 1H); ¹³C NMR(CDCl₃, 400 MHz) δ 177.0, 143.6, 135.9, 129.6, 128.5, 128.1, 128.0,127.8, 126.8, 69.0, 52.3; MS (MAB/HR/N2) 409.1271 HRMS calcd forC₂₇H₂₁O₂S 409.126227. found 409.127133.

EXAMPLE 7 3-Biphenyl-4-yl-2-tritylsulfanyl-propionic acid

¹H NMR (CDCl₃, 400 MHz) δ 11.40 (br s, 1H), 7.59–7.06 (m, 24H), 3.24(dd, 1H), 3.05 (dd, 1H), 2.60 (dd, 1H); ¹³C NMR (CDCl₃, 400 MHz) δ178.4, 144.0, 140.7, 139.7, 136.4, 129.6, 129.5, 128.7, 127.9, 127.1,127.0, 126.9, 68.5, 49.1, 38.7; MS (FAB-/NBA) 499.0.

EXAMPLE 8 3-Naphthalen-2-yl-2-tritylsulfanyl-propionic acid

¹H NMR (CDCl₃, 400 MHz) δ 11.50 (br s, 1H), 7.86–7.73 (m, 3H), 7.53–7.09(m, 19H), 3.33 (dd, 1H), 3.21 (dd, 1H), 2.75 (dd, 1H); ¹³C NMR (CDCl₃,400 MHz) δ 178.5, 144.0, 134.9, 133.3, 132.3, 129.6, 128.0, 127.8,127.6, 127.5, 127.2, 126.9, 126.0, 125.7, 68.6, 49.1, 39.2; MS(FAB-/NBA) 473.0.

EXAMPLE 9 3-(4-Fluoro-phenyl)-2-tritylsulfanyl-propionic acid

¹H NMR (CDCl₃, 400 MHz) δ 7.50 (d, 6H), 7.30–7.20 (m, 9H), 6.92–6.83 (m,4H), 3.08 (dd, 1H), 2.88 (dd, 1H), 2.41 (dd, 1H); ¹³C NMR (CDCl₃, 400MHz) δ 177.9, 143.8, 132.9, 130.6, 130.5, 129.5, 127.9, 126.9, 115.2,114.9, 95.7, 68.6, 49.0, 38.1; MS (FAB-/LR/NBA) 441.0.

EXAMPLE 10 3-(4-Methoxy-phenyl)-2-tritylsulfanyl-propionic acid

MS (FAB-/NBA) 453.0.

EXAMPLE 11 3-(4-Benzyloxy-phenyl)-2-tritylsulfanyl-propionic acid

¹H NMR (CDCl₃, 400 MHz) δ 7.50–7.20 (m, 21H), 6.89 (s, 3H), 5.02 (s,2H), 3.14 (dd, 1H), 2.92 (dd, 1H), 2.48 (dd, 1H); ¹³C NMR (CDCl₃, 400MHz) δ 178.7, 157.7, 144.1, 136.9, 130.2, 129.8, 129.7, 128.5, 128.0,127.9, 127.4, 127.0, 114.7, 69.9, 68.5, 49.4, 38.3; MS (FAB-/LR/NBA)528.8.

EXAMPLE 12 3-Cyclohexyl-2-tritylsulfanyl-propionic acid

MS (FAB-/LR/NBA) 429.0.

EXAMPLE 13 2-Tritylsulfanyl-propionic acid

MS (MAB/HR/N2) 348.1194 HRMS calcd for C₂₂H₂₀O₂S 348.118402. found348.119379.

EXAMPLE 14 2-Tritylsulfanyl-hexanoic acid

¹H NMR (CDCl₃, 400 MHz) δ 10.97 (br s, 1H), 7.58 (m, 6H), 7.37–7.21 (m,9H), 2.99 (dd, 1H), 1.80–1.74 (m, 1H), 1.43–1.21 (m, 5H), 0.89 (t, 3H);¹³C NMR (CDCl₃, 400 MHz) δ 179.6, 144.2, 129.6, 127.9, 126.8, 68.2,47.3, 32.7, 29.3, 22.1, 13.7; MS (FAB-/LR/NBA) 389.0.

EXAMPLE 15 4-Methyl-2-tritylsulfanyl-pentanoic acid

MS (thio FAB) 389.

EXAMPLE 16 3-Methyl-2-tritylsulfanyl-butyric acid

MS (FAB-/LR/NBA) 375.1.

EXAMPLE 17 3,3-Dimethyl-2-tritylsulfanyl-butyric acid

¹H NMR (CDCl₃, 400 MHz) δ 11.59 (br s, 1H), 7.56 (d, 6H), 7.33–7.18 (m,9H), 2.62 (s, 1H), 0.97 (s, 9H); MS (FAB-/NBA) 389.1.

EXAMPLE 18 3-tert-Butoxy-2-tritylsulfanyl-propionic acid

¹H NMR (CDCl₃, 400 MHz) δ 7.49 (d, 6H), 7.32–7.21 (m, 9H), 3.26 (t, 1H),3.16 (dd, 1H), 2.83 (dd, 1H), 1.02 (s, 9H); MS (MAB/HR/N2) 420.1755 HRMScalcd for C₂₆H₂₈O₃S 420.175917. found 420.175534.

EXAMPLE 19 3-tert-Butoxy-2-tritylsulfanyl-butyric acid

¹H NMR (CDCl₃, 400 MHz) δ 7.54–7.47 (m 6H), 7.33–7.20 (m, 9H), 3.23(m,1H), 2.96 (d, 1H), 1.19 (s, 9H), 0.99 (m, 3H); ¹³C NMR (CDCl₃, 400MHz) δ 172.4, 143.9, 129.5, 127.9, 126.8, 69.3, 68.6, 53.7, 28.5, 28.4,21.2; MS (FAB-/LR/NBA) 432.9.

EXAMPLE 20 3-(2-Carboxy-2-tritylsulfanyl-ethyl)-indole-1-carboxylic acidtert-butyl ester.

¹H NMR (CDCl3, 400 MHz) δ 7.45–7.12 (m, 20H), 3.27 (dd, 1H), 3.14 (dd,1H), 2.67 (dd, 1H), 1.65 (s, 9H); ¹³C NMR (CDCl₃, 400 MHz) δ 178.1,149.4, 143.9, 129.5, 127.8, 126.8, 124.3, 124.2, 122.3, 118.8, 116.3,115.0, 83.5, 68.5, 60.3, 47.4, 28.6, 28.1; MS (FAB-/NBA) 562.0.

EXAMPLE 21 2-Ethoxycarbonyl-succinic acid 4-tert-butyl ester 1-ethylester

Diethyl malonate (31.15 g, 194.48 mmoles) was first transferred at 0° C.to a stirring suspension of sodium hydride (4.67 g, 194.58 mmoles) indry THF (200 mL). To this resulting mixture, was added dropwise (at 0°C.) a solution of t-butyl bromoacetate (23.00 g, 117.91 mmoles).Formation of a white precipitate was instantly observed. The mixture wasthen stirred at room temperature until complete consumption of thestarting material (1.5 hours as confirmed by TLC: 20% ethyl acetate:80%hexanes). The solvent was then evaporated and the residue was taken-upin 500 mL diethyl ether. This organic phase was washed with water (3×300mL) and washed once with brine (200 mL) before being dried (anhydrousMgSO₄) and evaporated. The obtained residue was first purified bydistillation to remove excess malonate and then by flash chromatography(SiO₂, 15% ethyl acetate:85% hexanes) to afford 30.00 g (92.8% yield) ofthe desired compound as an oil: IR (film) 2982, 1733, 1258, 1152, 1037cm⁻¹; ¹³C NMR (CDCl₃, 100 MHz)

169.5, 168.1, 80.8, 61.2, 47.7, 33.9, 27.6.

EXAMPLE 22 2-Carboxy-succinic acid 4-tert-butyl ester

To a stirring solution of the compound of example 21 (20.00 g, 72.91mmoles) in ethanol (250 mL) was added a 10% aqueous solution ofpotassium hydroxide (9.65 g, 172.07 mmoles). The resulting mixture wasthen stirred at reflux for eight hours. When the reaction was completed(as confirmed by TLC: 20% ethyl acetate:80% hexanes), the mixture wasfirst cooled down and the solvent was evaporated. The obtained residuewas taken-up in water (200 mL) and washed with diethyl ether (2×200 mL)before being acidified with a 10% aqueous solution of hydrochloric acid.The product was then extracted from the aqueous phase with ethyl acetate(4×200 mL). The organic layers were combined, dried (anhydrous MgSO₄),filtered and evaporated under vacuum to yield 13.30 g (83.6% yield) ofthe desired diacid as an oil. This product was used directly in the nextreaction without further purification: ¹H NMR (CDCl₃, 400 MHz)

11.11 (br s, 2H), 3.81 (t, 1H), 2.87 (d, 2H), 1.40 (s, 9H); ¹³C NMR(CDCl₃, 100 MHz)

173.0, 169.7, 81.9, 47.3, 33.9, 27.7.

EXAMPLE 23 2-Methylene-succinic acid 4-tert-butyl ester

To a stirring solution of the compound of example 22 (13.30 g, 60.95mmoles) in ethanol (500 mL) at 0° C. were sequentially addeddiethylamine (11.17 g, 152.72 mmoles) and a 37% aqueous solution offormaldehyde (4.57 g, 152.18 mmoles). The resulting mixture was stirredat room temperature for 12 hours. The solvent was then directlyevaporated under vacuum and the obtained residue was dissolved in asaturated aqueous solution of sodium bicarbonate (400 mL). The aqueousphase was twice washed with ethyl acetate and then acidified with a 10%aqueous solution of hydrochloric acid. The aqueous layer was extractedwith ethyl acetate (4×400 mL) to afford, after concentration, 3.00 g(26.4% yield) of the desired decarboxylated product. This compound wasused directly in the next reaction without further purification: ¹H NMR(CDCl₃, 400 MHz)

6.41 (s, 1H), 5.78 (s, 1H), 3.25 (s, 2H), 1.43 (s, 9H); ¹³C NMR (CDCl₃,300 MHz)

171.6, 169.8, 133.6, 130.1, 81.2, 38.5, 27.8.

EXAMPLE 24 2-Acetylsulfanylmethyl-succinic acid 4-tert-butyl ester

To a stirring solution of the compound of example 23 (3.00 g, 16.11mmoles) in anhydrous THF (16 mL), was added drop wise thiolacetic acid(3.68 g, 48.34 mmoles). The mixture was stirred at room temperature for24 hours. The solvent was then evaporated under vacuum and the residuewas directly purified by flash chromatography (SiO₂, 20% ethylacetate:80% hexanes) to afford 1.80 g (42.6% yield) of the desiredproduct as a yellow oil. IR (film) 2980, 2652, 1729, 1255, 1152 cm⁻¹;HRMS (EI) Calcd for C₁₁H₁₉O₅S: 263.0953. Found: 263.0944.

EXAMPLE 25 2-Mercaptomethyl-succinic acid 4-tert-butyl ester

To a stirring solution of the crude compound of example 24 (6.87 g;26.19 mmoles) in degassed methanol (270 mL) at room temperature wasadded drop wise a 10% aqueous solution of potassium carbonate (7.24 g;52.38 mmoles). The mixture was stirred for three hours at roomtemperature. After complete consumption of the starting material (asconfirmed by TLC: 30% ethyl acetate-70% hexanes), the reaction mixturewas poured into degassed water (500 mL) and was extracted with degasseddichloromethane (2×200 mL) and then was acidified with a 10% aqueoushydrochloric acid solution. The aqueous layer was finally extracted withethyl acetate (4×500 mL). The organic extracts were combined, dried(anhydrous MgSO₄), filtered and evaporated to yield 8.10 g of the crudedesired compound. This residue was used directly in the next stepwithout any further purification.

EXAMPLE 26 2-Tritylsulfanylmethyl-succinic acid 4-tert-butyl ester

To a stirring solution of the compound of example 25 (8.10 g; 36.77mmoles) in dry THF (25 mL) at room temperature, was transferred dropwisea solution of triphenylmethyl chloride (12.30 g; 44.13 mmoles) in dryTHF (25 mL). The mixture was stirred at room temperature for 24 hours.The solvent was then evaporated under vacuum and the residue wasdirectly subjected to flash chromatography (SiO₂, gradient of 15% ethylacetate in hexanes to 50% ethyl acetate in hexanes) to give 3.50 g(20.6% yield) of the pure desired protected compound: ¹H NMR (CDCl₃, 400MHz)

7.50 (d, 6H), 7.29 (m, 9H), 2.58 (m, 5H), 1.45 (S, 9H); ¹³C NMR (CDCl₃,100 MHz) □ 179.31, 170.32, 144.34, 129.47, 127.93, 127.81, 126.75,81.03, 66.93, 40.73, 36.13, 32.66, 27.90.

EXAMPLE 27 5-Tritylsulfany-pentanoic acid ethyl ester

To a suspension of sodium hydride (989 mg, 24.47 mmol) in anhydrous DMF(60 mL) was added portion wise triphenylmethanethiol. After 15 minutesof stirring, ethyl 5-bromovalerate (2.8 mL, 17.48 mmol) was injected andthe mixture was stirred at ambient conditions for 24 h. The reactionmixture was portioned between ether and saturated ammonium chloride. Thewater phase was extracted several times with ether. The recombinedorganic layer was washed with brine, dried, filtered and evaporated. Thecrude was flashed using 5–10% ethyl acetate in hexanes. ¹H-NMR(CDCl₃,400 MH

: 1.23(t, J=7.0 Hz, 3H); 1.42(m, 2H); 1.59(m, 2H); 2.16(m, 4H); 4.09(q,J=7.0 Hz, 2H), 7.21–7.30, 7.42(m, 15H).

EXAMPLE 28 2-(3-Tritylsulfanyl-propyl)-succinic acid 4-tert-butyl ester1-ethyl ester

To a solution of LDA (2M, 9.1 mL, 18.17 mmol) at −70° C. was addedHMPA(10 mL) followed by the drop wise addition of a solution of thecompound of example 27 (6.12 g, 15.14 mmol) in anhydrous THF (10 mL).The reaction mixture was allowed to warm up to −40° C. and was stirredfor another 30 minutes, after which, a solution of tert-butylbromoacetate (2.5 mL, 16.65 mmol) in anhydrous THF (10 mL) was added.The reaction mixture was stirred for 1 h at the same temperature andthen allowed to warm up to room temperature followed by stirring for 24h. The reaction mixture was portioned between ether and saturatedammonium chloride. The water phase was extracted several times withether. The recombined organic layer was washed with brine, dried,filtered and evaporated. Silica gel chromatography using 5% ethylacetate in hexanes provided the product as a light yellow oil.¹H-NMR(CDCl₃, 400 MH

: 1.24(t, J=7.1 Hz, 3H); 1.43(s, 9H); 1.40–1.57(m, 4H); 2.15(m, 2H);2.24(m, 1H); 2.58(m, 2H); 4.12(q, J=7.1 Hz, 2H), 7.19–7.23, 7.26–7.30,7.41(m, 15H).

EXAMPLE 29 2-(3-Tritylsulfanyl-propyl)-succinic acid 4-tert-butyl ester

To a solution of the compound of example 28 (3.00 g, 5.78 mmol) inmethanol (30 mL) and water (10 mL) was added NaOH (1N, 11.6 mL, 11.6mmol). The mixture was stirred vigorously under ambient conditions.After 24 hours, most of the methanol has evaporated. The residue wasportioned between water and ether. The water phase was extracted twotimes with ether, then acidified with HCl (1N) to pH 1. The water phasewas extracted three times with ethyl acetate. The recombined organicphase was dried over sodium sulfate, filtered, and evaporated. Theresidue was flashed using 5% methanol in chloroform to give the productas a white solid. ¹H-NMR(CDCl₃, 400 MH

: 1.45(s, 9H); 1.35–1.64(m, 4H); 1.58(m, 1H); 2.16(m, 2H); 2.26(m, 1H);2.64(m, 1H); 4.12(q, J=7.1 Hz, 2H), 7.19–7.29, 7.40(m, 15H).

EXAMPLE 30 2-Hydroxymethyl-acrylic acid ethyl ester

To a mixture of triethylphosphonoacetate (44.80 g, 0.20 mol) and a 37%aqueous solution of formaldehyde (59.95 mL, 0.80 mol) stirred at roomtemperature was slowly added a saturated solution of potassium carbonate(48.4 g, 0.35 mol). At the end of the addition, the temperature reached30–35° C. and stirring was continued for an additional 2 hours. Themixture was extracted with ether (3×100 ml) and the combined organicextracts were dried (anhydrous MgSO₄) and the solvent was evaporated invacuo. The remaining oil was purified by flash chromatography (SiO₂, 15%ethyl acetate−85% hexanes) to afford 18.42 g (70.8%) of the desiredcompound as an oil: ¹H NMR (CDCl₃, 400 MHz) δ 1.19 (t, 3H), 3.50 (s,1H), 8.85 (q, 2H), 5.7, 6.31 (s, 1H); ¹³C NMR (CDCl₃, 400 MHz)

166.7, 140.1, 125.3, 61.1, 53.9, 14.4.

EXAMPLE 31 2-Bromomethyl-acrylic acid ethyl ester

Phosphorus tribromide (17.16 g, 63.41 mmol) was added to a stirredsolution of the compound of example 30 (17.92 g, 138.1 mmol) in dryether (132 mL) at −10° C. The temperature was allowed to rise to 20° C.and stirring was continued for 3h. Water (80 mL) was then added at −10°C. and the mixture was extracted with hexane (3×45 mL). The organicextracts were washed with saturated sodium chloride solution (2×45 mL)and dried with anhydrous MgSO₄. Evaporation of solvent under vacuum gave15.07 g (yield 56.6%) of the crude product, which was used directly inthe next reaction without any further purification. ¹H NMR (CDCl₃, 400MHz) δ 6.25 (s, 1H), 5.88 (s, 1H), 4.2 (q, 2H), 4.11 (s, 2H), 1.25 (t,3H); ¹³C NMR (CDCl₃, 400 MHz)

166.7, 140.1, 125.3, 61.1, 53.9, 14.4.

EXAMPLE 32 2-tert-Butoxycarbonyl-4-methylene-pentanedioic acid1-tert-butyl ester 5-ethyl ester

Di-tert-butylmalonate (14.95 g, 77.50 mmol) was transferred at 0° C. toa suspension of sodium hydride (3.1 g, 77.50 mmol) in dry THF (78 mL).To this resulting mixture was added dropwise at 0° C., a solution of thecompound of example 31 (14.96 g, 77.50 mmol) in dry THF (78 mL). Themixture was then stirred at room temperature until complete consumptionof the starting material (30 minutes as confirmed by TLC: 15% hexane-85%ethyl acetate). The solvent was then evaporated and the residue wastaken-up into 400 mL of water followed by extraction with ether (4×300ml). The organic extracts were combined and washed with brine. Dryingand evaporation of the solvent gave the crude product. Purification byflash chromatography (SiO₂, 10% hexane-90% ethyl acetate) afforded 22.62g (89% yield) of the desired compound as an oil: ¹H NMR (CDCl₃, 300 MHz)δ 6.21 (s, 1H, CH), 5.62 (s, 1H), 4.25 (q, 2H), 3.51 (t, 1H), 2.83 (d,2H), 1.45 (s, 18H), 1.31 (t, 3H).

EXAMPLE 33 2-tert-Butoxycarbonyl-4-tritylsulfanylmethyl-pentanedioicacid 1-tert-butyl ester 5-ethyl ester

Triphenylmethanethiol (20.58 g, 74.5 mmol) in dry THF (40 mL) was firstadded drop wise at 0° C. to a suspension of sodium hydride (3.00 g, 74.5mmol) in dry THF (60 mL). After the addition was complete, the mixturewas stirred for 15 min at 0° C. followed by 45 minutes at roomtemperature. To the resulting suspension, the compound of example 32(20.58 g, 62.4 mmol) dissolved in dry THF (40 ml) was added drop wise at0° C. The mixture was then stirred at room temperature until completeconsumption of the starting material (2 hours as confirmed by TLC: 97%hexane-3% ethyl acetate). The crude product was isolated as in example32 and was then purified by flash chromatography (SiO₂, 5% hexane-95%ethyl acetate) to afford 28.28 g (75.5% yield) of the desired compoundas an oil: ¹H NMR (CDCl₃, 400 MHz) δ 7.31 (m, 15H), 4.17 (q, 2H), 3.03(m, 1H), 2.49 (m, 1H), 2.42 (m, 1H), 2.31 (m, 2H), 2.21 (m, 1H), 1.44(s, 18H), 1.29 (t, 3H); ¹³C NMR (CDCl₃, 400 MHz)

176.3, 167.9, 144.4, 129.4, 127.7, 126.5, 81.5, 66.6, 60.6, 51.4, 42.6,33.6, 30.4, 27.7, 14.1.

EXAMPLE 34 2-tert-Butoxycarbonyl-4-tritylsulfanylmethyl-pentanedioicacid 1-tert-butyl ester

To a stirred solution of the ester of example 33 (2.1 g, 3.31 mmol) inethanol (11 mL) was added 17.4 ml of a 10% aqueous solution of potassiumhydroxide. The resulting mixture was stirred for 24 hours. Afterevaporation of the solvent, the obtained residue was taken-up into 50 mlof water and washed with diethyl ether (2×50 mL). The aqueous layer wasthen acidified to pH 3 with a 10% aqueous solution of hydrochloric acidat 0° C. and immediately extracted with ethyl acetate (5×50 ml). Theorganic extracts were combined, washed with water, and dried overanhydrous magnesium sulfate. Evaporation under vacuum gave the crudeproduct which was purified by flash chromatography (SiO₂, 70% hexane-30%ethyl acetate) to afford 880 mg of the desired compound: ¹H NMR (CDCl₃,400 MHz) δ 7.31 (m, 15H), 3.04 (t, 1H), 2.95 (m, 1H), 2.25 (m, 2H), 1.85(m, 1H), 1.44 (s, 18H); ¹³C NMR (CDCl₃, 400 MHz)

173.9, 168.5, 144.9, 129.7, 128.3, 128.0, 82.0, 51.9, 43.3, 33.7, 30,5,27.2.

EXAMPLE 35 2-(3′,4′-Dimethoxy-biphenyl-4-yl)-ethylamine

To a solution of 4-bromophenethylamine (10 g, 48.98 mmol) in anhydrousDMF (150 mL), containing anhydrous triethylamine (35 mL, 244.9 mol), wasadded Boc₂O. The reaction mixture was heated for 15 minutes at 50° C.After cooling to room temperature, brine (100 mL) and HCl (1N, 100 mL)were added subsequently, and the mixture was extracted several timeswith ether. The recombined organic layer was washed again with brine,dried over sodium sulfate, filtered and evaporated. The crude wasflashed with 10% ethyl acetate in hexanes to give the[2-(4-bromo-phenyl)-ethyl]-carbamic acid tert-butyl ester. ¹H NMR(CDCl₃, 300 MH

: 1.41(s, 9H); 2.22(t, J=7.1 Hz, 2H); 3.31(m, 2H); 4.67(s, broad, 1H);7.03(d, J=8.2 Hz, 2H); 7.38(d, J=8.2 Hz, 2H).

A mixture of the above mentioned compound (1.00 g, 3.33 mmol),3,4-dimethoxyphenylboronic acid (1.21 g, 6.66 mmol), and potassiumhydroxide (2N, 5 mL, 10 mmol) in THF (15 mL) was degassed using argonfor 5 minutes. Palladium tetrakis-triphenylphosphine (200 mg, 0.167mmol) was added and the mixture was heated at 85° C. After 24 hours, thereaction mixture was allowed to cool to room temperature. Brine (20 mL)was added and the reaction mixture was extracted several times withether. The recombined organic phase was extracted with brine, dried oversodium sulfate, filtered, and evaporated. Silica gel chromatography ofthe crude using 20% ethyl acetate/n-hexane afforded[2-(3′,4′-Dimethoxy-biphenyl-4yl)-ethylamine]-carbamic acid tert-butylester as a white solid. ¹H-NMR (CDCl₃, 300 MH

: 1.44(s, 9H); 2.82(t, J=6.9 Hz, 2H); 3.39(m, 2H); 3.91(s, 3H); 3.93(s,3H); 4.67(s, broad, 1H); 6.92(d, J=8.2 Hz, 1H); 7.11(m, 2H); 7.25(m,2H); 7.48(d, J=8.1 Hz, 2H).

A solution of the above mentioned compound (1.14 g, 3.19 mmol) inanhydrous methanol (50 mL) was cooled in ice bath and then treated dropwise with acetyl chloride. Stirring was continued for 30 minutes at thesame temperature followed by overnight stirring at room temperature.About 30 mL of the solvent was removed by evaporation and the mixturewas diluted with 200 mL of ether. The entitled product was collected asa white solid by filtration, followed by washing with anhydrous etherand drying under high vacuum. ¹H-NMR (D₂O, 300 MH

: 2.80(t, J=6.9 Hz, 2H); 3.40(m, 2H); 3.91(s, 3H); 3.93(s, 3H); 4.77(s,broad, 1H); 7.00(d, J=8.2 Hz, 1H); 7.15(m, 2H); 7.26(m, 2H); 7.50(d,J=8.1 Hz, 2H).

The amines of examples 36–42 were prepared by the Suzuki cross couplingmethod as illustrated above in example 35.

EXAMPLE 36 4′-(2-Amino-ethyl)-biphenyl-4-carbonitrile EXAMPLE 372-(4-Pyridin-2-yl-phenyl)-ethylamine EXAMPLE 38(4′-(2-Amino-ethyl)-biphenyl-4-ol Example 39[4′-(2-Amino-ethyl)-biphenyl-4-yl]-dimethyl-amine EXAMPLE 402-(3′,4′-Dimethoxy-biphenyl-4yl)-ethylamine EXAMPLE 412-(4′-Phenoxy-biphenyl-4-yl)-ethylamine EXAMPLE 422-(4′-Methoxy-biphenyl-4-yl)-ethylamine

The following amines were prepared by reduction of the correspondingcommercially available nitrites:

EXAMPLE 43 2-Naphthalen-1-yl-ethylamine EXAMPLE 442-Naphthalen-2-yl-ethylamine EXAMPLE 45 2-(3-Phenoxy-phenyl)-ethylamineEXAMPLE 46 2,2-Diphenyl-ethylamine EXAMPLE 47 4-Phenyl-butyric acidmethyl ester

A solution of 4-phenyl-butyric acid (10 g, 60.29 mmol) in methanol (200mL) was prepared. To this solution was added hafnium chloride-THFcomplex (0.6 g, 1.2 mmol) and the mixture was stirred overnight at roomtemperature. The solvent was evaporated and the residue was partitionedbetween water and diethyl ether. The aqueous phase was extracted againwith ether. The recombined organic layer was extracted with saturatedsodium hydrogen carbonate, brine, and water. The solvent was evaporatedto dryness, after drying over sodium sulfate and filtration. Theresultant oily product was used directly in the next reaction withoutany further purification. ¹H-NMR (CDCl₃, 300 MH

: 1.97(p, J=7.5 Hz, 2H); 2.35(3, J=7.4 Hz, 2H); 2.66(t, J=7.4 Hz, 2H);3.67(s, 3H); 7.22(m, 5H).

EXAMPLE 48 2-Phenethyl-succinic acid 4-tert-butyl ester 1-methyl ester

A solution of 4-phenyl-butyric acid methyl ester (compound of example47) (5 g, 28.05 mmol) in anhydrous THF (200 mL) was prepared and cooledto −78° C. To this solution was added by cannula a freshly preparedsolution of LDA (28.05 mmol, ca. 1M) in anhydrous THF. The reactionmixture was stirred for 30 minutes, then treated drop wise with a cooledsolution of tert-butyl bromoacetate (4.6 mL, 30.86 mmol) in anhydrousTHF (30 mL). HMPA (1.2 mL) was added and stirring was continued at −78°C. for an additional 30 minutes followed by slowly warming to roomtemperature. Stirring was continued overnight. Saturated ammoniumchloride was added and after stirring, the organic phase was separated.The aqueous phase was extracted several times with ethyl acetate. Therecombined organic layer was washed with brine, dried over sodiumsulfate and filtered. The solvent was evaporated and the crude productwas flashed using 10% ethyl acetate in hexanes. Based on its ¹H NMRspectrum, the flashed oily colorless product was considered sufficientlypure for the next step. ¹H-NMR(CDCl₃, 300 MH

: 1.43(s, 9H); 1.99(m, 2H); 2.40(m, 1H); 2.73(m, 4H); 2.84(m, 1H);3.71(s, 3H); 7.21(m, 5H).

EXAMPLE 49 2-Phenethyl-succinic acid 1-methyl ester

A solution of the compound of example 48 in dichloromethane (20 mL) andcontaining about 1 mL of water was prepared. The solution was cooled inan ice bath and treated with TFA (10 mL). The reaction mixture wasstirred for 1 hour while in the ice bath, followed by 1 hour at roomtemperature. The solvent was evaporated and the residue was partitionedbetween water and dichloromethane. The aqueous phase was extracted twicewith dichloromethane. The recombined organic layer was washed withbrine, dried over sodium sulfate, filtered and evaporated. The crude oilwas flashed using 10% ethyl acetate in hexanes followed by 2% methanolin chloroform to give the product as a colorless oil. ¹H-NMR(CDCl₃, 300MH

: 1.92(m, 2H); 3.70 (s, 3H); 2.62(m, 4H); 2.87(m, 1H); 3.72(s, 3H).

EXAMPLE 50 4-(4-Benzyl-2-oxo-oxazolidin-3-yl)-4-oxo-2-phenethyl-butyricacid methyl ester

To a solution of the compound of example 49 (2.51 g, 10.62 mmol) inanhydrous THF (60 mL) and containing triethyl amine (1.8 mL, 12.74 mmol)at −70° C. was added drop wise trimethylacetyl chloride. After 15minutes, the reaction mixture was placed in an ice bath and stirred for45 minutes after which time, the reaction mixture was cooled to −78° C.In a separate flask, n-butyl lithium (2.5M in hexanes, 43 mL, 10.62mmol) was added to a solution of R-4-benzyl-2-oxazolidinone in anhydrousTHF at −78° C. After 15 minutes of stirring, this mixture wastransferred by cannula to the former solution. The resultant mixture wasstirred for 20 minutes at −78° C. followed by an additional 2 h in anice bath. The reaction was quenched by the addition of a saturatedsolution of ammonium chloride. The mixture was extracted several timesusing ethyl acetate. The recombined organic layer was extracted withbrine, saturated sodium hydrogen carbonate, brine, and water,respectively. The solvent was evaporated following drying over sodiumsulfate and filtration. The resultant crude material was flashed using30% ethyl acetate in hexanes to give the product as a white solid.¹H-NMR(CDCl₃, 300 MH

: 1.89(m, 1H); 2.05(m, 1H); 2.75(m, 3H); 3.00(m, 1H); 3.68(m, 3H);3.75(s, 3H); 4.18(m, 2H); 4.65(m, 1H); 7.21(m, 5H), 7.33(m, 5H).

EXAMPLE 513-(4-Benzyl-2-oxo-oxazolidine-3-carbonyl)-2-phenethyl-pentanedioic acid5-tert-butyl ester 1-methyl ester

A solution of the compound of example 50 (2.40 g, 6.07 mmol) inanhydrous THF (60 mL) was cooled to −78° C. A solution of NaHDMS in THF(1M, 6.7 mmol, 6.7 mL) was injected over 10 minutes. The reactionmixture was stirred for an additional 20 minutes, after which time, asolution of tert-butyl bromoacetate in anhydrous THF (5 mL) was slowlyinjected. After stirring for 1 h at −78° C., and 3 h at −48° C., thereaction was quenched by the addition of a saturated ammonium chloridesolution. The mixture was extracted several times using ethyl acetate.The recombined organic layer was extracted successively with brine,saturated sodium hydrogen carbonate, brine and water. The solvent wasevaporated following drying over sodium sulfate and filtration. Theresultant crude material was flashed using 20% ethyl acetate in hexanesto give the product as a white solid. ¹H-NMR(CDCl₃, 300 MH

: 1.40, 1.41(2s, 9H); 1.74–1.92(m, 5H); 2.45–2.95(m, 4H); 3.31(m, 1H);3.69, 3.73(2 s, 3H); 4.11(m, 2H); 4.50, 4.66(2 m, 1H); 7.25(m, 10H).

EXAMPLE 52 3-Benzyloxycarbonyl-2-phenethyl-pentanedioic acid5-tert-butyl ester 1-methyl ester

To a stirred solution of benzyl alcohol (0.5 mL, 4.41 mmol) in anhydrousTHF (16 mL) at −70° C., was added n-butyl lithium (2.5M, 1.4 mL, 3.53mmol). After 10 minutes of stirring, the solution was transferred bymeans of a cannula to a solution of the compound of example 51 inanhydrous THF (15 mL) at −70° C. The reaction was allowed to warm to−10° C. over a period of 2 h, after which time, it was placed in an icebath and stirred for another 50 minutes. Finally, the reaction wasquenched by the addition of a saturated ammonium chloride solution. Themixture was extracted several times using ethyl acetate. The recombinedorganic layer was extracted with brine, dried over sodium sulfate,filtered and evaporated. The resultant crude was flashed using 10% ethylacetate in hexanes to give the product as a colorless oil. ¹H-NMR(CDCl₃,300 MH

: 1.39, 1.40(2 s, 9H); 1.70(m, 1H); 2.01(m, 1H); 2.31–2.74(m, 4H);2.83(m, 1H); 3.21(m, 1H); 3.61, 3.64(2s, 3H); 5.11(m, 2H); 7.08–7.35(m,10H).

EXAMPLE 53 3-Carboxy-2-phenethyl-pentandioic acid 5-tert-butyl ester1-methyl ester

A solution of the compound of example 52 (700 mg, 1.59 mmol) in ethanol(95%, 100 mL) was hydrogenated in the presence of a catalytic amountpalladium (10% on charcoal). After 24 h of stirring, the reaction wascomplete. The mixture was filtered over a celite pad and the solvent wasevaporated. The residue was distributed between ethyl acetate and water.The aqueous phase was extracted twice more with ethyl acetate. Therecombined organic layer was washed with brine, dried over sodiumsulfate, filtered, and evaporated.

EXAMPLE 54 3-(2-Biphenyl-4-yl-ethylcarbamoyl)-2-phenethyl-pentanedioicacid 5-tertbutyl ester 1-methyl ester

A solution of the compound of example 53 (590 mg, 1.68 mmol) and HOBt(272 mg, 2.016 mmol) in anhydrous DMF was stirred in an ice bath for 10minutes. Biphenyl ethylamine (404 mg, 2.02 mmol) was added followed bythe addition of DIC (320 μL, 2.01 mmol). After 20 minutes, the ice bathwas removed and stirring was continued at room temperature. After 24 hof stirring, the reaction mixture was partitioned between ethyl acetateand HCl (1M, excess). The aqueous phase was extracted several times withethyl acetate. The recombined organic layer was extracted with brine,saturated sodium hydrogen carbonate, and brine. The solvent wasevaporated following drying over sodium sulfate and filtration. Thecrude was flashed using 25% EtOAc in hexanes. ¹H-NMR(CDCl₃, 300 MH

: 1.41(s, 9H); 1.82(m, 2H); 2.23(m, 1H); 2.55(t, J=8.1 Hz, 2H); 2.75(m,5H); 3.54(m, 1H); 3.70(s, 3H); 6.09(t, J=5.7 Hz, 1H); 7.13–7.36,7.41–7.49, 7.55–7.58(m, 14H).

EXAMPLE 55 3-(Biphenyl-4-yl-ethylcarbamoyl)-2-phenethyl-pentanedioicacid 5-tert-butyl ester

A solution of the compound of example 54 (344 mg, 0.65 mmol) in methanolwas placed in an ice bath and treated slowly with a solution of sodiumhydroxide (1N, 1.3 mL, 1.3 mmol). After finishing the addition, thereaction mixture was stirred for 24 h at room temperature. The solventwas removed and the residue was portioned between ether and water. Afterseparation, the aqueous phase was acidified using HCl (1N) to pH 1 andextracted three times with ethyl acetate. The recombined organic phasewas dried over sodium sulfate, filtered, and evaporated to give theproduct as colorless oil.

EXAMPLE 56N-[1-tert-Butoxycarbonyl-2-(1H-indol-3-yl)-ethyl]-3-(9H-fluoren-9-ylmethoxycarbonylamino)-succinamicacid tert-butyl ester

To H-Trp-OBut (2.00 g, 6.75 mmol) in DMF (50 mL) was added sequentiallyFmoc-Asp(OBut)-OH (2.78 g, 6.75 mmol), HOBt (0.91 g, 6.75 mmol), BOP(2.99 g, 6.75 mmol) and DIPEA (2.35 mL, 13.50 mmol) at room temperature.The mixture was stirred for 2 h at room temperature. The reactionmixture was then diluted with ethyl acetate (300 mL) and washed withdiluted KHSO₄ (5%), NaHCO₃ (5%), H₂O, and brine. Drying (Na₂SO₄) andconcentration under vacuum gave the title dipeptide, 4.2 g (97%), as awhite foam which was pure as indicated by its spectroscopic data: ¹H NMR(CDCl₃, 300 MHz) d 7.90 (br s, 1H), 7.71 (br d, 2H), 7.57 (br d, 1H),7.50 (t, 1H), 7.49–7.0 (m, 8H), 5.80 (d, 1H), 4.7 (q, 1H), 4.61–4.02 (m,4H), 3.22 (m, 2H), 2.8 (dd, 1H), 2.55 (dd, 1H), 1.39 (s, 9H), 1.27 (s,9H); ¹³C NMR (CDCl₃, 75 MHz) d 170.2, 169.8, 143.6, 141.1, 135.8, 127.6,127.5, 127.0, 125.0, 122.6, 122.0, 119.9, 119.3, 118.8, 110.9, 110.1,81.9, 81.6, 67.1, 60.3, 53.5, 50.9, 46.9, 27.9, 27.7, 27.3, 14.1; MS(PosFAB) 653, 654 (MH)+.

EXAMPLE 573-Amino-N-[1-tert-butoxycarbonyl-2-(1H-indol-3-yl)-ethyl]-succinamicacid tert-butyl ester

This compound was prepared by Fmoc deprotection of the compound ofExample 56 using diethylamine in dichloromethane. Isolation by flashchromatography gave the title compound (73%): ¹H NMR (CDCl₃, 300 MHz) d8.42 (br s, 1H), 7.88 (d, 1H), 7.62 (d, 1H), 7.42–7.11 (m, 3H), 4.81(dt, 1H), 3.57 (dd, 1H), 3.32 (m, 2H), 2.70 (dd, 1H), 2.32 (dd, 1H),1.43 (s, 9H), 1.38 (s, 9H).

EXAMPLE 58 2-Ethoxycarbonyl-succinic acid 4-tert-butyl ester

To the tri-ester of example 21 (38 g, 0.14 mol) in ethanol (456 mL) wasadded 77.6 mL of a 10% aqueous solution of potassium hydroxide. Theresulting mixture was refluxed for 3 hours. The isolated product waspurified by flash chromatography (SiO₂, 90% dichloromethane-10%methanol) to afford 20.68 g (60.6% yield) of the title compound: ¹H NMR(CDCl₃, 400 MHz)

4.16 (q, 2H), 3.72 (t, 1H), 2.80 (d, 2H), 1.35 (s, 9H), 1.20 (t, 3H);¹³C NMR (CDCl₃, 400 MHz)

170.0, 168.9, 81.6, 62.4, 47.7, 34.5, 28.2, 14.5.

EXAMPLE 59 2-tert-Butoxycarbamoyl-succinic acid 4-tert-butyl ester1-ethyl ester

To a stirring solution of O-tert-butyl hydroxylamine hydrochloride (3.67g, 29.3 mmol) in dry THF (30 mL) at room temperature was addeddiisopropylethyl amine (3.60 g, 27.9 mmol). To the resulting mixture wastransferred the compound of example 58 (6.87 g, 27.9 mmol), as asolution in dry THF (30 mL), followed by HOBt (5.35 g, 27.9 mmol). Themixture was cooled to 0° C. and EDC (5.35 g, 27.9 mmol) was added in oneportion. The reaction mixture was then allowed to warm to roomtemperature and was stirred for 24 h. The solvent was evaporated and theresidue was taken-up into 500 ml of ethyl acetate and subsequentlywashed with a 5% aqueous solution of KHSO₄ (2×200 mL), with a 5% aqueoussolution of NaHCO₃ (2×200 mL), and finally with water. After drying(anhydrous MgSO₄) and evaporation, the crude product was purified byflash chromatography (SiO₂, 60% hexane-40% ethyl acetate) to afford 5.93g (66.0% yield) of the desired product: ¹H NMR (CDCl₃, 400 MHz)

8.61 (s, 1H), 4.20 (q, 2H), 3.55 (t, 1H), 2.93 (d, 3H), 1.43 (s, 9H),1.28 (s, 9H); ¹³C NMR (CDCl₃, 400 MHz)

195.8, 171.2, 169.7, 83.1, 81.9, 62.4, 46.8, 34.1, 28.4, 26.6, 14.5.

EXAMPLE 60 2-tert-Butoxycarbamoyl-succinic acid 4-tert-butyl ester

To 2-tert-butoxycarbamoyl-succinic acid 4-tert-butyl ester 1-ethyl ester(5.92 g, 18.6 mmol) in ethanol (224 mL) was added 20.91 mL of a 10%aqueous solution of potassium hydroxide. The resulting mixture was thenstirred at room temperature for 3 hours and then worked up. The crudeproduct was purified by flash chromatography (SiO₂, 90%dichloromethane-10% methanol) to afford 3.06 g (57.0% yield) of thedesired mono acid: 1H NMR (CDCl3, 400 MHz)

3.65 (t, 1H), 2.78 (dd, 3H), 1.45 (s, 9H), 1.25 s, 9H); 13C NMR (CDCl3,400 MHz)

171.2, 169.7, 84.0, 82.0, 46.8, 34.5, 28.5, 26.5.

EXAMPLE 61 3-Methoxycarbonyl-pentanedioic acid mono-tert-butyl ester

A solution of anhydrous diisopropylamine (22.3 mL, 158.9 mmol) in dryTHF (250 mL) was prepared. To this solution at −78° C. was added dropwise n-butyl lithium (2.5 M in n-hexane, 66.6 mL, 166.5 mmol). Stirringwas continued at the same temperature for an additional 15 minutesfollowed by 20 minutes at room temperature. The reaction mixture wasnext cooled to −78° C. and was transferred via cannula to a solution ofsuccinic acid monomethyl ester (10 g, 75.7 mmol) in dry THF (100 mL) at−78° C. HMPA (3.3 mL, 18.9 mmol) was added, followed, after about 10minutes, with the slow introduction of a solution of tert-butylbromoacetate (11.2 mL, 75.7 mmol) in dry THF (100 mL). After 30 minutesat −78° C., the reaction mixture was allowed to warm up and stirring wascontinued for 48 hours at room temperature. The crude reaction mixturewas then distributed between ethyl acetate (500 mL), HCl (1N, excess),and brine (200 mL). The aqueous layer was extracted several times withfresh ethyl acetate. The recombined organic extracts were washed withbrine, dried over sodium sulfate, filtered, and evaporated. Purificationby flash chromatography (SiO₂, 100% CHCl₃ to 2% methanol in CHCl₃)afforded the product (12.5 g, 67%) as a light yellow oil: ¹H NMR(CDCl₃,400 MHz)

: 3.67 (s, 3H), 3.18 (m, 1H), 2.47–2.82 (m, 4H), 1.49 (s, 9H).

EXAMPLE 62 2-(Trityloxycarbamoyl-methyl) succinic acid 4-tert-butylester 1-methyl ester

A solution of the compound of example 61 (10 g, 40.6 mmol) in anhydrousDMF (50 mL) was successively treated with HOBt (5.5 g, 40.6 mmol) andO-trityl hydroxalamine (11.2 g, 40.6 mmol). The reaction mixture wasstirred first in an ice bath for 15 minutes and then treated with DIC(6.4 mL, 40.6 mmol). After stirring for 20 minutes, the ice bath wasremoved and the reaction mixture was stirred for 24 hours at roomtemperature. The reaction mixture was distributed between ethyl acetate(200 mL) and a mixture of potassium hydrogen sulfate (1N, 45 mL, 1.5 eq)and brine (excess). The aqueous phase was extracted several times withfresh ethyl acetate. The combined organic extracts were washed withbrine, sodium hydrogen carbonate (10%, excess), brine, and finally driedover sodium sulfate. Evaporation and then purification by flashchromatography (SiO₂, 20% to 30% of ethyl acetate/n-hexane) afforded theproduct (12.8, 63%) as a white solid: ¹H NMR (CDCl₃, 400 MHz) □ 7.71 (d,1H), 7.31–7.45 (m, 15H), 3.63 (s, 3H), 2.95 (m, 1H), 2.05–2.40 (m, 4H),1.40 (s, 9H).

EXAMPLE 63 2-(Trityloxycarbamoyl-methyl) succinic acid 4-tert-butylester

A solution of the diester of example 62 (10.5 g, 20.8 mmol) in methanol(100 mL) was cooled in an ice bath and was treated drop wise with asolution of sodium hydroxide (1N, 42 mL). Stirring was continued for 30minutes and then for an additional 48 hours at room temperature. Themethanol was evaporated in a rotary evaporator and the alkaline reactionmixture was diluted with water followed by extracting with ether. The pHwas adjusted to 4 by the addition of HCl (2N) and the acidic solutionwas extracted several times with ethyl acetate. The combined organicextracts were washed with brine, dried over sodium sulfate, filtered andevaporated to give the desired product (10.2, 100%) as a white foam: ¹HNMR (CDCl₃, 400 MHz)

7.70 (d, 1H), 7.26–7.46 (m, 16H), 2.72–2.99 (m, 1H), 1.99–2.42 (m, 4H),1.42 (s, 9H).

EXAMPLE 64 3-Methoxycarbonyl-hexanedioic acid 1-tert-butyl ester

To a solution of n-BuLi (2.5M, 2.6 mL, 71.5 mmol) in anhydrous THF (200mL) was added anhydrous diisopropyl amine (9.6 mL, 68.30 mmol) at −78°C. After 20 minutes of stirring, a solution of monomethyl glutarate (5g, 32.5 mmol) in anhydrous THF (20 mL) was added drop wise undercontrolled temperature. After 30 minutes, a solution of tert-butylbromoacetate (4.9 mL, 32.50 mmol) in anhydrous THF (20 mL) was slowlyadded followed by the addition of HMPA (8 mL). The reaction mixture wasstirred at −78° C. for an additional 2 h followed by 24 h at roomtemperature. The reaction mixture was partitioned between HCl (2N,excess) and ethyl acetate. The organic phase was separated and theaqueous phase was extracted several times with ethyl acetate. Therecombined organic layer was extracted with brine, dried over sodiumsulfate, filtered, and evaporated. The residue was first flashed withchloroform and then with 2–5% methanol in chloroform. The product wasobtained as a light yellow oil. ¹H-NMR(CDCl₃, 400 MH

: 1.42(s, 9H); 1.89(m, 2H); 2.24(m, 2H); 2.64(m, 2H); 2.84(m, 1H);3.69(s, 3H).

EXAMPLE 65 2-(2-Trityloxycarbamoyl-ethyl-succinic acid 4-tert-butylester 1-methyl ester

To a solution of the compound of example 64 (1.10 g, 4.19 mmol) and HOBt(0.70 g, 5.03 mmol) in DMF (5 mL) were added O-trityl hydroxylamine(1.82 g, 6.28 mmol) and DIC (800 μL), respectively. The reaction mixturewas stirred for 24 h at room temperature. The reaction mixture waspartitioned between HCl (1N, excess) and ethyl acetate. The aqueousphase was extracted several times with ethyl acetate. The recombinedorganic layer was washed with brine, saturated sodium bicarbonate, andbrine. The solvent was evaporated following drying over sodium sulfate,and the crude was flashed with 20–30% ethyl acetate in hexanes.¹H-NMR(CDCl₃, 400 MH

: 1.42(s, 9H); 1.58, 2.14, 2.47(3 m, 7H); 3.69(s, 3H); 7.34(m, 15H);7.88(d, broad, 1H).

EXAMPLE 66 2-(2-Trityloxycarbamoyl-ethyl)-succinic acid 4-tert-butylester

To a solution of the compound of example 65 (2.27 g, 4.39 mmol) in amixture of methanol/water (4:1, 50 mL) was added a solution of sodiumhydroxide (1N, 9 mL). The resulting mixture was stirred for 24 h at roomtemperature. The solvent was evaporated and the residue was partitionedbetween water and ether. The alkaline phase acidified using HCl (6N) andextracted with ethyl acetate followed by drying over sodium sulfate,filtration and evaporation. The product was converted to an off-whitefoam under high vacuum. ¹H-NMR(CDCl₃, 400 MH

: 1.42(s, 9H); 1.59(m, 3H); 1.88–2.54(m, 4H); 7.33(m, 15H); 7.46(s,broad, 1H).

EXAMPLE 67 4-(4-Benzyl-2-oxo-oxazolidin-3-yl)-4-oxo-butyric acid ethylester

A solution of R-(+)-4-benzyl-2-oxazolidine (25 g, 144.3 mmol) inanhydrous THF (800 mL) was cooled to −70° C. and treated drop wise withn-BuLi (2.5M in hexanes, 52.5 mL, 131.2 mmol). After 20 minutes ofstirring, ethyl 4-chloro-4-oxobutyrate (neat, 23.5 mL, 131.2 mmol) wasadded drop wise. The reaction mixture was stirred for 15 minutes at −70°C., followed by 1 h in an ice bath. The reaction was quenched by theaddition of a saturated ammonium chloride solution. The organic layerwas extracted three times with ethyl acetate. The recombined organiclayer was extracted with a saturated sodium bicarbonate solution, brineand water. After drying over sodium sulfate and filtration, the solventwas evaporated and the crude product was flashed using 20–40% ethylacetate/hexanes. The product was obtained as a white solid.¹H-NMR(CDCl₃, 400 MH

: 1.27(t, J=7.1, 3H); 2.71(m, 3H); 3.26(m, 3H); 4.18(m, 4H); 4.6(m, 1H);7.19–7.36(m 5H).

EXAMPLE 68 4-(4-Benzyl-2-oxo-oxazolidine-3-carbonyl)-pentanedioic acidtert-butyl ester ethyl ester

A solution of the compound of example 67 (5.0 g, 16.38 mmol) inanhydrous THF (160 mL) was cooled to −78° C. A solution of NaHDMS in THF(1M, 16.4 mL, 16.4 mmol) was injected over 10 minutes. The reactionmixture was stirred for an additional 20 minutes, after which time, asolution of tert-butyl bromoacetate in anhydrous THF (10 mL) was slowlyinjected. After stirring for 1 h at −78° C. and 3 h at −48° C., thereaction was quenched by the addition of a saturated ammonium chloridesolution. The mixture was extracted several times with ethyl acetate.The recombined organic layer was successively extracted with brine, asaturated sodium hydrogen carbonate solution, brine and water. Thesolvent was evaporated following drying over sodium sulfate andfiltration. The resultant crude product was flashed with 20% ethylacetate in hexanes to give the product as a white solid. ¹H-NMR(CDCl₃,400 MH

: 1.24(t, J=7.2, 3H); 1.44(s, 9H); 2.54(m, 2H); 2.77(m, 3H); 3.31(m,1H); 4.14(m, 4H); 4.43(m, 1H); 4.69(m, 1H); 7.26(m 5H).

EXAMPLE 69 3-Benzyloxycarbonyl-pentandioic acid tert-butyl ester ethylester

To a stirred solution of benzyl alcohol (1.3 mL, 12.3 mmol) in anhydrousTHF (50 mL) at −70° C., was added n-butyl lithium (2.5M, 4 mL, 10 mmol).After 10 minutes of stirring, the solution was transferred via cannulato a solution of example 65 (3.44 g, 8.20 mmol) in anhydrous THF (50 mL)at −70° C. The reaction was allowed to warm to −10° C. over a period of2 h, after which time, it was placed in an ice bath and stirred foranother 50 minutes. Finally, the reaction was quenched by the additionof a saturated ammonium chloride solution. The mixture was extractedseveral times with ethyl acetate. The recombined organic layer wasextracted with brine, dried over sodium sulfate, filtered andevaporated. The resultant crude product was flashed with 10% ethylacetate in hexanes to give the product as a colorless oil. ¹H-NMR(CDCl₃,400 MH

: 1.21(t, J=7.2, 3H); 1.41(s, 9H); 2.49–2.86(m, 4H); 3.28(q, J=6.9 Hz,1H); 4.13(m, 2H); 5.1(m, 2H); 7.32(m 5H).

EXAMPLE 70 3-Carboxy-pentanedioic acid tert-butyl ester ethyl ester

A solution of the compound of example 69 (2.29 g, 6.54 mmol) in ethanol(95%, 500 mL) was hydrogenated in the presence of a catalytic amount ofpalladium (10% on charcoal). After 24 h of stirring, the reaction wascomplete. The mixture was filtered over a celite pad and the solventevaporated. The residue was distributed between ethyl acetate and water.The aqueous phase was extracted twice more with ethyl acetate. Therecombined organic layer was washed with brine, dried over sodiumsulfate, filtered, and evaporated. The product was sufficiently pure onthe basis of its ¹H-NMR spectrum to be used in the next step.¹H-NMR(CDCl₃, 400 MH

: 1.23(t, J=7.1, 3H); 1.41(s, 9H); 2.53–2.81(m, 4H); 3.25(m, 1H);4.13(m, 2H).

EXAMPLE 71 3-(2-Biphenyl-4-yl-ethylcarbamoyl)-pentanedioic acidtert-butyl ester ethyl ester

A solution of the compound of example 70 (1.55 g, 5.96 mmol) inanhydrous DMF (5 mL) was treated successively with DIEA (5.2 mL),4-biphenylethyl amine (2.40 g, 11.92 mmol) and TBTU (2.76 g, 8.34 mmol).The reaction mixture was stirred for 24 h at room temperature. Thereaction mixture was treated with HCl (1M, excess) and the mixture wasextracted several times with ethyl acetate. The recombined organic layerwas washed with brine, dried over sodium sulfate, filtered andevaporated. The residue was flashed with 30% ethyl acetate in hexanesand the desired product was obtained as a white solid. ¹H-NMR(CDCl₃, 400MH

: 1.22(t, J=7.2, 3H); 1.42(s, 9H); 2.32–2.42(m, 2H); 2.60–2.75(m, 2H);2.83(t, J=7.1 Hz, 2H); 3.02(m, 1H); 3.53(m, 2H); 4.11(m, 2H); 6.25(m, t,5.1 Hz, 1H); 7.30(m, 3H); 7.43(m, 2H); 7.57(m, 4H).

EXAMPLE: 72 3-(2-Biphenyl-4-yl-ethylcarbamoyl)-pentandioic acidmono-tert-butyl ester

To a solution of the compound of example 71 (1.09 g, 2.48 mmol) inmethanol (50 mL) was added drop wise a sodium hydroxide solution (1N, 5mL, 5 mmol). After stirring the reaction mixture for 24 h, the solventwas evaporated. The residue was partitioned between water and ether. Theaqueous phase was acidified with concentrated HCl, and then extractedwith ethyl acetate. The organic phase was washed with brine, dried,filtered and evaporated to give the product as a white solid.1H-NMR(CDCl3, 400 MH

: 1.37(s, 9H); 2.28–2.61(m, 4H); 2.79(t, J=7.3 Hz, 2H); 3.08(m, 1H);3.27(m, 1H); 3.39 (m, 2H); 6.25(m, t, 5.1 Hz, 1H); 7.26(m, 3H); 7.36(m,2H); 7.52(m, 4H).

EXAMPLE 73 4-(Bis-benzyloxy-phosphoryl)-butyric acid ethyl ester

To a suspension of NaH (60%, 2.52 g, 63 mmol) in DMF (60 mL) was addeddibenzyl phosphite (12 mL, 48.45 mmol). After 15 minutes of stirring,ethyl-4-bromobutyrate was added and the mixture was stirred for 48 h atroom temperature. The DMF was removed by evaporation. The residue waspartitioned between brine, ammonium chloride and ether. The water phasewas extracted several times with ether. The recombined organic layer wasextracted with brine, dried over sodium sulfate, filtered andevaporated. The residue was flashed using 50% ethyl acetate in hexanesto afford the product as a colorless oil. ¹H-NMR(CDCl₃, 400 MH

: 1.23(t, J=7.2 Hz, 3H); 1.77–2.04(m, 4H); 2.37(m, 2H); 4.10(q, J=7.2Hz, 2H), 5.03(m, 4H); 7.31(m, 10H). ³¹P-NMR(CDCl₃, 400 MHz): 32.78.

EXAMPLE 74 4-(Bis-benzyloxy-phosphoryl)-butyric acid

A solution of the compound of example 73 (8.66 g, 23.02 mmol) in THF(150 mL) was treated drop wise with a solution of lithium hydroxide-monohydrate (1.97 g, 46.04 mmol) in water (150 mL). The reaction wascomplete after about 1 h, according to TLC analysis. Most of the solventwas evaporated. The residue was partitioned between water and ether. Theorganic phase was separated and the alkaline phase was extracted againwith ether. It was next acidified with HCl (6M) to pH˜1 followed byseveral extractions with ethyl acetate. The recombined organic layer wasdried over sodium sulfate, filtered, and evaporated to give the productas colorless oil. 1H-NMR(CDCl3, 400 MH

: 1.88(m, 4H); 2.40(t, J=6.9 Hz, 2H); 4.97,4.96, 5.05(2dd, J1=11.8 Hz.J2=8.1 Hz, 4H); 7.35(m, 10H). 31P-NMR(CDCl3, 400 MHz): 33.15.

EXAMPLE 75 [4-(4-Benzyl-2-oxo-oxazolidin-3-yl)-4-oxo-butyl]-phosphonicacid dibenzyl ester

To a stirred solution of the compound of example 74 (6.45 g, 18.52 mmol)in anhydrous THF (100 mL) was added triethyl amine. The reaction mixturewas then cooled to −70° C. After 10 minutes, trimethyl acetyl chloride(2.5 mL, 20.37 mmol) was added and stirring was continued for another 15minutes at −70° C. followed by 1 h in an ice bath. In a separate flask,n-BuLi (7.5 mL) was added drop wise to a solution ofR-4-benzyl-2-oxazolidine in anhydrous THF (60 mL) at −70° C. After 15minutes, this mixture was transferred by cannula to the former solutioncooled to −70° C. Stirring was continued for 20 minutes at −70° C.,followed by 2 h in an ice bath. A saturated ammonium chloride solutionwas added (excess), and the mixture was extracted several times withethyl acetate. The recombined organic layer was washed with brine, witha saturated sodium bicarbonate solution and brine, followed by dryingover sodium sulfate, filtration and evaporation. The residue was flashedstarting with 40% ethyl acetate, followed by 60% ethyl acetate inhexanes. The product was obtained as a white solid. 1H-NMR(CDCl3, 400 MH

: 1.84–2.04(, 4H); 2.70(m, 1H); 2.86(m, 1H); 3.03(m, 1H), 3.24(m, 1H);4.15(m, 2H); 4.63(m, 1H); 4.99(m, 4H); 7.32(m, 15H). 31P-NMR(CDCl3, 400MHz): 33.23. FAB:508.1 (MH+).

EXAMPLE 763-(4-Benzyl-2-oxo-oxazolidine-3-carbonyl)-5-(bis-benzyloxy-phosphoryl)-pentanoicacid tert-butyl ester

A solution of the compound of example 75 (5.42 g, 10.70 mmol) inanhydrous THF (100 mL) was prepared and cooled to −70° C. A solution ofNaHDMS in THF (1M, 40.2 mL, 40.2 mmol) was added drop wise. The reactionmixture was stirred for 1 h at −70° C. and for 4 h at −48° C., afterwhich time, the reaction was not complete according to TLC analysis. Thereaction was allowed to stir for another 1 h at −30° C. At this stage,the completion of the reaction could not yet be confirmed by TLCanalysis. The reaction was quenched by the addition of a saturatedsolution of ammonium chloride. The mixture was extracted several timeswith ethyl acetate. The recombined organic layer was washed with brine,filtered and evaporated. The residue was flashed using 50% ethyl acetatein hexanes and the product was obtained as a colorless oil.¹H-NMR(CDCl₃, 400 MH

: 1.41(s, 9H); 1.83(m, 3H); 2.34(m, 1H); 2.71(m, 2H); 3.29(m, 1H);3.15(m, 2H); 4.59(m, 1H); 4.94, 5.05(2m, 4H); 7.17–7.37(m, 15H). ³¹P-NMR(CDCl₃, 400 MHz): 33.23.

EXAMPLE 77 2[2-(Bis-benzyloxy-phosphoryl)-ethyl]-succinic acid4-tert-butyl ester

A solution of the compound of example 76 (1.03 g, 1.66 mmol) in amixture of THF/water (4:1, 20 mL) was cooled in an ice bath and treateddrop wise with a hydrogen peroxide solution (30%, 0.7 mL). After 5minutes, a solution of lithium hydroxide monohydrate (1M, 6.6 mL, 6.6mmol) was added over a period of 10 minutes. The reaction was completedfollowing 2 h of stirring in the ice bath. At this stage, an aqueoussolution of sodium sulfite (850 mg, 6.64 mmol, 5 mL) was added. The icebath was removed and the reaction mixture was stirred for another 30minutes. Most of the solvent was removed by evaporation and the residuewas extracted with ether. The alkaline phase was acidified with HCl (6M)to pH˜1 and extracted three times with ethyl acetate. The recombinedorganic layer was washed with brine, dried over sodium sulfate, filteredand evaporated. The desired product was obtained as a colorless oil.¹H-NMR (CDCl₃, 400 MH

: 1.40(s, 9H); 1.85(m, 4H); 2.27, 2.58(2dd, J₁=16.5 Hz. J₂=5.6 Hz, 4H);2.84(m, 1H); 4.98(m, 4H); 7.31(m, 10H). ³¹P-NMR (CDCl₃, 400 MHz): 33.14.

EXAMPLE 78 2-(Diethoxy-phosphoryl)-4-phenyl-butyric acid tert-butylester

Diethylphosphonoacetate (1.00 g, 3.96 mmol) was added drop wise at 0° C.to a stirring suspension of sodium hydride (0.095 g, 3.96 mmol) inanhydrous THF (50 mL). The mixture was then allowed to stir for 10minutes at 0° C. and then for 1 hour at room temperature before it wascooled again to 0° C. followed by the addition of phenethyl bromide(0.73 g, 3.96 mmol). The mixture was then allowed to proceed for 24hours at room temperature. The solvent was evaporated and the residuewas taken-up in water and extracted 4 times with ethyl acetate. Theorganic extracts were combined, washed with water and brine, dried usingmagnesium sulfate, filtered and evaporated. The crude residue waspurified by flash chromatography (ethyl acetate in hexanes, 10%–50%) toafford the desired compound as a colorless oil. ¹H NMR (CDCl₃, 400 MHz)δ 7.18 (m, 2H), 7.09 (m, 2H), 4.07–3.99 (m, 4H), 2.82–2.72 (m, 1H),2.69–2.60 (m, 1H), 2.54–2.45 (m, 1H), 2.25–2.12 (m, 1H), 2.08–1.94 (m,1H), 1.41 (s, 9H), 1.24–1.18 (m, 6H); ¹³C (CDCl₃, 400 MHz) δ 167.7,140.4, 128.3, 128.2, 125.9, 81.5, 62.2, 46.3, 45.0, 34.1, 28.5, 27.7,16.1; ³¹P (CDCl₃, 400 MHz) δ 23.7. HRMS calcd. for C₁₈H₂₉O₅P 356.1753.found 356.1757.

EXAMPLE 79 2-(Diethoxy-phosphoryl)-4-phenyl-butyric acid

Trifluoroacetic acid (4 mL) was added drop wise to a stirring solutionof 2-(diethoxy-phosphoryl)-4-phenyl-butyric acid tert-butyl ester (1.00g, 2.81 mmol) in anhydrous dichloromethane (16 ml). The mixture wasstirred for 2 hours at room temperature followed by the removal of thesolvent. The crude residue was purified by flash chromatography (100%dichloromethane-2.5% methanol in dichloromethane) to afford the desiredcompound as a yellowish oil. ¹H NMR (CDCl₃, 400 MHz) δ 11.21 (br s, 1H),7.29–7.14 (m, 5H), 4.24–4.12 (m, 4H), 3.09–3.00 (m, 1H), 2.82–2.75 (m,1H), 2.64–2.57 (m, 1H), 2.35–2.27 (m, 1H), 2.15–2.11 (m, 1H), 1.31–1.25(m, 6H); ¹³C (CDCl₃, 100 MHz) δ 170.4, 140.4, 128.4, 128.3, 126.1, 63.4,63.2, 45.1, 43.8, 34.0, 28.5, 16.0; ³¹p (CDCl₃, 400 MHz) δ 24.3; HRMScalcd. for C₁₄H₂₁O₅P 300.1127. found 300.1129.

EXAMPLE 80 (4-Phenyl-butyl)-phosphinic acid

To a solution of NaH₂PO₂.H₂O (2.65 g, 25 mmol) and 4-phenyl 1-butene(1.5 mL, 10 mmol) in methanol (40 mL) was added triethylborane (10 mL,10 mmol, 1.0 M solution in hexanes). The clear colorless solution wasstirred at room temperature for a period of 4 hours, with the reactionvessel open to the atmosphere. The reaction mixture was diluted withethyl acetate (100 mL) and washed with an aqueous KHSO₄ solution (100mL, 2.0 M). The aqueous layer was extracted three times with ethylacetate and the combined organic extracts were dried (Na₂SO₄) andconcentrated to provide the crude product (1.78 g) as a pale yellow oilwhich was carried on to the next step without any further purification.³¹P NMR (CDCl₃) δ 37.5.

EXAMPLE 81 (4-Phenyl-butyl)-phosphinic acid benzyl ester

To a solution of the compound of example 80 (1.75 g, 8.83 mmol) in dryCH2Cl2 (40 mL) was added benzyl alcohol (1.83 mL, 17.7 mmol), DMAP (108mg, 0.88 mmol) and EDC (1.91 g, 10.0 mmol). The mixture was then stirredat room temperature for 18 h. The mixture was concentrated in vacuum,taken-up in ethyl acetate and washed with NaHCO3 (5%) and H2O. Drying(Na2SO4) and concentration gave 2.8 g of a pale yellow oil which waspurified by flash chromatography (SiO₂, ethyl acetate:hexane (1:1)) togive 1.92 g of the title compound.

EXAMPLE 82N-[1-Benzyloxycarbonyl-2-(1H-indol-3-yl)-ethyl]-3-tert-butoxycarbonylamino-succinamic acid benzyl ester

To a commercially available solution of H₂N-Trp-OBn (3.15 g, 9.76 mmol)in dry DMF (50 mL) was added neat DIPEA (1.7 mL, 9.76 mmol). Theslightly yellow solution was then cooled to 0° C. and Boc-Asp (OBn) OSuwas added in one portion. After 10 min at 0° C., the ice bath wasremoved and the mixture was allowed to warm to room temperature followedby an additional 5 hours of stirring. The reaction mixture was treatedas previously described in example 56. Drying (Na₂SO₄) and concentrationunder vacuum gave the title dipeptide (5.46 g; 96%) as white foam: m.p:46–48° C., R_(f) (ethyl acetate)=0.32. The spectroscopic data wasconsistent with the structure.

EXAMPLE 833-Amino-N-[1-benzyloxycarbonyl-2-(1H-indol-3-yl)-ethyl]-succinamic acidbenzyl ester

To a round-bottomed flask (1L) was added a solution of (HCl) dioxane(180 mL. 4.0 M). This solution was cooled to 0° C. followed by theaddition of the compound of example 82 in one portion while stirring.The ice bath was removed and the reaction was further stirred for 30 minat room temperature before it was concentration under vacuum at <25° C.Washing the residue with dry ether gave the product (4.39 g) as a tannedsolid. This product was used for the next reactions without furtherpurification.

EXAMPLE 84 3-(2-Biphenyl-4-yl-ethylcarbamoyl)-6-tritylsulfanyl-hexanoicacid tert-butyl ester

To a solution of the compound of example 29 (200 mg, 0.41 mmol) and HOBt(66 mg, 0.49 mmol) in DMF (2 mL), were added 4-biphenylethylamine (124mg, 0.62 mmol) and DIC (78 μL) respectively. The reaction mixture wasstirred for 24 h at room temperature. The reaction mixture waspartitioned between HCl (0.5N, excess) and ethyl acetate. The aqueousphase was extracted several times with ethyl acetate. The recombinedorganic layer was washed with brine, with a saturated sodium hydrogencarbonate solution, and again with brine. The solvent was evaporatedfollowing drying over sodium sulfate, and the crude was flashed using30% ethyl acetate in hexanes. ¹H-NMR (CDCl₃, 400 MH

: 1.55(s, 9H); 1.41–1.57(m, 2H); 2.04–2.28(m, 6H); 2.54(m, 1H); 2.80(m,2H); 3.45–3.55(m, 2H), 5.66(m, 1H); 7.19–7.57(m, 24H).

EXAMPLE 85 3-(2-Biphenyl-4-yl-ethylcarbamoyl)-6-mercapto-hexanoic acid

This compound was obtained by standard deprotection of the compound ofexample 84, followed by HPLC purification.

EXAMPLE 863-{2-[3-tert-Butoxycarbonyl-2-(2-tritylsulfanyl-acetylamino)-propionylamino]-2-carboxy-ethyl}-indole-1-carboxylic acid tert-butyl ester

To Fmoc-Asp (O^(t)But)-OH (987 mg, 2.40 mmol) in1-methyl-2-pyrrolidinone (NMP, 10 mL) was added HOBT (324 mg) and theclear colorless solution was cooled to 0° C. Diisopropyl carbodiimide(DIC, 375 μL, 2.4 mmol) was then added drop wise. After the addition wascomplete, the clear solution was left to warm to room temperature andwas further stirred for 30 minutes. Concurrently, H-Trp (Boc)-2-Cl-Trtresin (1.0 g, 0.4 mmol) was swelled in just enough NMP for 30 min. Theactive ester solution was then cannulated to the solid phase peptidesynthesis vessel, containing the resin, and the mixture was gentlystirred at room temperature for 2 hours. The mixture was then filteredand the resin was washed with DMF (3×), DCM (3×), MeOH (3×), DCM (3×),and finally with MeOH (3×). At this stage, the resin had a loading of0.32 mmol/g. The Fmoc group was removed using 25% piperidine in DMF. Toaccess the purity, a small portion (20 mg) of the resin was cleaved togive H-Asp (OBut)-Trp(Boc)-OH with purity exceeding 98% according to NMRand HPLC. ¹H NMR (CD₃OD, 400 MHz) δ 8.06 (d, 1H), 7.7 (d, 1H), 7.51 (s,1H), 7.25 (dt, 2H), 4.6 (dd, 1H), 3.94 (dd, 1H), 3.12 (dd, 1H), 2.95(dd, 1H), 2.65 (dd, 1H), 1.67 (s, 9H), 1.45 (s, 9H).

The rest of the resin was coupled to S-trityl thioglycolic acid (6equivalents) in the same manner as mentioned above. After 2 hours atroom temperature, the resin was filtered, washed, dried and cleavedusing cocktail B (DCM:HFIP, 4:1) to give the title compound (200 mg, 85%based on initial loading of resin) as a white foam: ¹H NMR (CD₃OD, 300MHz) δ 8.14 (d, 1H), 7.60–7.15 (m, 20H), 4.75 (m, 1H), 4.40 (m, 1H),3.30 (dd, 1H), 3.16 (dd, 1H), 3.0 (dd, 2H), 2.60 (dd, 1H), 2.30 (dd,1H), 1.67 (s, 9H), 1.42 (s, 9H); ¹³C NMR (CDCl₃, 300 MHz) δ 175.1,171.5, 170.8, 169.7, 150.1, 144.2, 135.7, 130.7, 129.9, 128.5, 127.5,125.0, 124.8, 123.2, 119.3, 115.7, 115.2, 84.3, 82.5, 68.3, 53.9, 53.1,49.5, 36.9, 36.0, 28.6, 28.2; MS (PosFAB) 814.1 (MH)⁺, HRMS calcd forC₄₅H₄₉O₈N₃SNa (MNa⁺) 814.3139. found 814.3157.

EXAMPLE 87N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-acetylamino)-succinamicacid

The fully protected peptide of example 86 was subjected to cleavagecocktail A to remove the protecting groups, and to liberate the crudetarget peptidic thioglycolylamide which was isolated as mentioned above(general procedures) and purified by preparative HPLC (49% overallyield): ¹H NMR (CD₃OD, 300 MHz) δ 7.55 (d, 1H), 7.32 (d, 1H), 7.14–6.98(m, 3H), 4.72 (ddd, 2H), 3.35 (dd, 1H), 3.31 (dd, 1H), 2.79 (dd, 1H),2.65 (dd, 1H); ¹³C NMR (CDCl₃, 300 MHz) δ 172.1, 171.3, 170.6, 169.8,135.3, 126.3, 122.1, 119.8, 117.3, 116.9, 109.7, 107.9, 52.0, 48.6,33.7, 25.6, 25.4; MS (Pos. FAB) 394.1 (MH)⁺

The following non-limiting examples wherein the □-substituent (P1substituent) is the variable, were prepared in a manner analogous tothat presented in Examples 86 and 87. Thus, the resin-bound dipeptideH₂N-Asp(O^(t)But)-Trp (Boc)-O-2-Cl-Tritylchloride resin (example 86) wascoupled to various substituted-2-tritylsulfanyl-propionic acidderivatives (Examples 4 to 20) using the coupling,cleavage/deprotection, purification protocols as previously described,to provide the following derivatives of which the preparation of thederivative carrying a phenmethyl □-substituent is described in detail.

EXAMPLE 883-{2-[3-tert-Butoxycarbonyl-2-(3-phenyl-2-tritylsulfanyl-propionylamino)-propionylamino]-2-carboxyethyl}-indole-1-carboxylicacid tert-butyl ester

This compound was prepared using the same protocol mentioned above forexample 86. Thus, the resin-bound dipeptide, H₂N-Asp(O^(t)But)-Trp(Boc)-O-2-Cl-Tritylchloride resin (example 86) was coupled to3-phenyl-2-tritylsulfanyl-propionic acid (example 4) using the DIC/HOBTmethod. To ensure complete coupling, the reaction was repeated with 2equivalents of the S-trityl acid derivative. After standard washing anddrying, the resin was cleaved using cocktail B to give the titlecompound as a crispy white foam: ¹H NMR (CD₃OD, 400 MHz) δ 8.12 (d, 1H),6.91–7.52 (m, 25H), 4.66 (t(d), 1H), 4.20 (t(d), 1H), 3.25 (dd, 1H),3.08 (dd, 1H), 2.92 (m, 2H), 2.61 (dd, 1H), 2.59 (dd, 1H), 2.12 (dd,1H), 1.67 (s, 9H), 1.35 (s, 9H).

EXAMPLE 89N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-3-phenyl-propionylamino)-succinamic acid

The fully protected peptide obtained above was subjected to cleavagecocktail A to remove the protecting groups and liberate the crude targetpeptide thioglycolylamide, which was isolated as mentioned above(general procedures), and purified by preparative HPLC: MS (Neg. FAB,NBA) 481.9 (M−2H).

EXAMPLE 90N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-propionylamino)-succinamicacid

MS (Pos. FAB) 508 (MH⁺).

EXAMPLE 91N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-4-methyl-pentanoylamino)-succinamicacid

MS (Pos. FAB) 534 (MH⁺).

EXAMPLE 92N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-3-methyl-butyrylamino)-succinamicacid

MS (Pos. FAB) 436 (MH⁺).

EXAMPLE 93N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(3-hydroxy-2-mercapto-propionylamino)-succinamicacid

MS (Pos. FAB) 424 (MH⁺).

EXAMPLE 94N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(3-hydroxy-2-mercapto-butyrylamino)-succinamicacid

MS (Pos. FAB) 438 (MH⁺), 460 (MNa⁺).

EXAMPLE 95N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-hexanoylamino)-succinamicacid

MS (Pos. FAB) 450 (MH⁺).

EXAMPLE 96N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-4-phenyl-butyrylamino)-succinamicacid

MS (Pos. FAB) 498 (MH⁺)

EXAMPLE 97N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-2-phenyl-acetylamino)-succinamicacid

MS (Pos. FAB) 470 (MH⁺).

EXAMPLE 983-(3-Biphenyl-4-yl-2-mercapto-propionylamino)-N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-succinamicacid

MS (Pos. FAB) 560 (MH⁺).

EXAMPLE 993-(3-(4-Benzyloxy-phenyl)-2-mercapto-propionylamino)-N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-succinamicacid

MS (Pos. FAB) 590 (MH⁺).

EXAMPLE 100N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-[3-(4-fluoro-phenyl)-2-mercapto-propionylamino]-succinamicacid

MS (Pos. FAB) 502 (MH⁺).

EXAMPLE 101N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-[2-mercapto-3-(4-methoxy-phenyl)-propionylamino]-succinamicacid

MS (Pos. FAB) 534 (MH⁺).

EXAMPLE 102N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(3-cyclohexyl-2-mercapto-propionylamino)-succinamicacid

MS (Pos. FAB) 490 (MH⁺).

EXAMPLE 103N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-[3-(1H-indol-3-yl)-2-mercapto-propionylamino]-succinamicacid

MS (Pos. FAB) 502 (MH⁺).

EXAMPLE 104N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-3-naphthalen-2-yl-propionylamino]-succinamicacid

MS (Pos. FAB) 534 (MH⁺).

The following non-limiting examples wherein the P′2 subsistent is thevariable, were prepared in a manner analogous to that presented inExamples 86 and 88. Thus, in each case, the required amino acid (oramino alcohol) was first loaded onto the 2-Cl-Trt resin followed by Fmocdeprotection, coupling with Fmoc-Asp (OBut)-OH, Fmoc deprotection andfinally coupling with (S)-3-phenyl-2-tritylsulfanyl-propionic acid.Employing the same cleavage/deprotection, and purification protocols aspreviously described, provided the following non-limiting examples ofwhich the preparation of the naphthyl derivative is described in detail.

EXAMPLE 105N-(1-Carboxy-2-naphthalen-2-yl-ethyl)-3-(3-phenyl-2-tritylsulfanyl-propionylamino)-succinamicacid tert-butyl ester

This compound was assembled by first loading the first amino acid,Fmoc-2-Nal-OH onto 2-Cl-Trt resin. Thus, to Fmoc-2-Nal-OH (0.26 g, 0.59mmol) in dry DCM (5 mL) was added diisopropylethylamine (311 □L, 1.78mmol) drop wise at room temperature. This solution was then added dropwise to the 2-Cl-Trt resin (0.50 g, 0.54 mmol, pre-swelled in dry DCMfor 30 minutes). After the addition was complete, the reaction mixturewas gently stirred at room temperature for 90 minutes. HPLC grademethanol (1 mL) was then added and gentle stirring continued for 10 moreminutes. The resin was filtered and washed with DCM (3×), DMF (3×),2-propanol (3×), DMF (3×), DCM (3×) and MeOH (3×), and dried to give 0.7g of the Fmoc-2-Nal-O-2Cl-Trt resin as yellow granules. At this stagethe resin loading was determined to be 0.7 mmol/g.

After removal of the Fmoc groups, Fmoc-Asp(OBut)-OH was coupled toH-2-Nal-O-2Cl-Trt-resin in the same manner mentioned above for example86. After removal of the Asp Fmoc group followed by the standard washingand drying of the resin, a small portion (15 mg) was cleaved to giveH-Asp (OBut)-2-Nal-OH with purity exceeding 98% according to NMR andHPLC: ¹H NMR (CD₃OD, 400 MHz) δ 7.95 (m, 3H), 7.71 (s, 1H), 7.42 (m,3H), 4.62 (dd, 1H), 3.97 (dd, 1H), 3.42 (dd, 1H), 3.15 (dd, 1H), 2.92(dd, 1H), 2.64 (dd, 1H), 1.45 (s, 9H).

The rest of the resin was coupled to(s)-3-phenyl-2-tritylsulfanyl-propionic acid as mentioned above (Example4) using the DIC/HOBT method. After isolation and cleavage usingcocktail B the title compound was obtained (262 mg, 87% yield based onthe initial loading of the resin) as a white foam: ¹H NMR (CD₃OD, 400MHz) δ 7.71 (m, 5H), 7.4 (m, 4H), 6.8–7.32 (m, 18H), 4.65 (m, 1H), 4.18(m, 1H), 3.31 (dd, 1H), 3.05 (dd, 1H), 2.88 (m, 2H), 2.57 (m, 2H), 2.1(dd, 1H).

EXAMPLE 106 N-(1-Carboxy-2-naphthalen-2-yl-ethyl)-3-(2-mercapto-3-phenylpropionylamino)-succinamic acid

The fully protected peptide obtained above was subjected to cocktail Ato remove the protecting groups and liberate the crude target peptidicthioglycolylamide which was isolated as mentioned above (generalprocedures) and purified by preparative HPLC (45% overall yield): ¹H NMR(CD₃OD, 300 MHz) δ 7.82 (m, 4H), 7.41 (m, 3H), 7.15 (m, 5H), 6.98 (ddd,2H), 3.48 (t, 1H), 3.31 (dd, 1H), 3.15 (dd, 1H), 3.03 (dd, 1H), 2.76 (m,2H), 2.63 (dd, 1H); ¹³C NMR (CDCl₃, 300 MHz) δ 171.4, 176.5, 176.1,174.8, 141.9, 138.0, 137.3, 136.3, 132.6, 131.8, 131.5, 131.4, 131.2,131.0, 130.1, 129.4, 129.0, 57.5, 53.7, 46.7, 45.4, 44.7, 40.8, 38.9; MS(Neg FAB, NBA) 492.9 (M−2H).

EXAMPLE 107N-(1-Carboxy-2-hydroxy-ethyl)-3-(2-mercapto-3-phenyl-propionylamino)-succinamic acid

MS (Pos. FAB) 385 (MH+), 407 (MNa+).

EXAMPLE 108N-[1-Carboxy-2-(4-hydroxy-phenyl)-ethyl]-3-(2-mercapto-3-phenyl-propionylamino)-succinamicacid

¹H NMR (MeOD-4, 400 MHz) □ 7.32–7.12 (m, 5H), 7.05 (d, 1H), 6.73 (d,1H), 4.70 (dd, 1H), 4.55 (dd, 1H), 3.61 (t, 1H), 3.19 (dd, 1H), 3.07(dd, 1H), 2.95 (m, 2H), 2.78 (dd, 1H), 2.65 (dd, 1H); ¹³C NMR (CDCl₃,100 MHz) □ 175.2, 173.4, 173.1, 172.2, 157.2, 139.1, 131.2, 130.3,129.2, 128.1, 127.5, 115.8, 55.2, 51.3, 44.2, 42.1, 37.6, 36.6. MS (Pos.FAB) 461 (MH+).

EXAMPLE 109N-[1-Carboxy-2-phenyl-ethyl)-3-(2-mercapto-3-phenyl-propionylamino)-succinamic acid

¹H NMR (MeOD-4, 400 MHz) □ 7.32–7.05 (m, 10H), 4.70 (dd, 1H), 4.59 (dd,1H), 3.61 (t, 1H), 3.15 (m, 2H), 2.92 (dd, 1H), 2.85 (dd, 1H), 2.78 (dd,1H), 2.63 (dd, 1H); ¹³C NMR (CDCl₃, 100 MHz) □ 175.1, 173.4, 173.1,172.2, 138.7, 137.8, 130.2, 130.1, 129.2, 127.4, 55.1, 51.9, 44.4, 42.8,38.1, 36.1. MS (Pos FAB) 445 (MH+)

EXAMPLE 110N-(2-Biphenyl-4-yl-1-Carboxy-ethyl)-3-(2-mercapto-3-phenyl-propionylamino)-succinamic acid

MS (Neg. FAB) 518(M+−2H); (65%).

EXAMPLE 111N-(1-Benzyl-2-hydroxy-ethyl)-3-(2-mercapto-3-phenyl-propionylamino)-succinamic acid

MS (Pos. FAB) 431(MH)+; (82%).

Following the same solid phase protocols as described above, thefollowing non-limiting examples wherein the P′1 L-Asp of the compound ofExample 31 has been replaced with D-Asp, L-Glu, or L-Gla, were prepared.

EXAMPLE 112N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-3-phenyl-propionylamino)-succinamicacid. (83%). EXAMPLE 1132-[2-[1-Carboxy-2-(1H-indol-3-yl)-ethylcarbamoyl]-2-(2-mercapto-3-phenyl-propionylamino)-ethyl]-malonicacid

(Pos. FAB) 542.7 (MH+); (45%)

EXAMPLE 1144-[1-Carboxy-2-(1H-indol-3-yl)-ethylcarbamoyl]-4-(2-mercapto-3-phenyl-propionylamino)-ethyl]-butyricacid (75%)

The preferred method for preparing the following compounds in which thetryptophan residue (Examples 87 and 89) has either been completelyreplaced by a variety of amine moieties, or only its carboxyl functionhas been transformed to amides and esters, involves fragmentcondensation strategy. Thus, after assembly of the desired peptide onthe solid support, cleavage from the resin was conducted using cocktailB as in Example 86 (see also the section of general procedures) in orderto only liberate the carboxy terminus for further elaboration. Example115, illustrating the preparation of the methyl amide, is arepresentative example.

EXAMPLE 115N-[2-(1H-indol-3-yl)-methylcarbamoyl-ethyl]-3-(2-mercapto-acetylamino)-succinamic acid

To a solution of the protected dipeptide of Example 86 (100 mg, 0.13mmol), methylamine hydrochloride (8.5 mg, 0.13 mmol), and HOBt (17 mg,0.13 mmol) in DMF (2 mL), was added EDCI (24 mg, 0.13 mmol) at 0° C.,followed by NMM (14 μL, 0.13 mmol) under argon. The ice bath was removedand the mixture was further stirred at room temperature for 16 hfollowed by the addition of an aqueous NaHCO₃ (5%) solution. The mixturewas extracted with EtOAc and the combined organic extracts weresequentially washed with NaHCO₃ (5%), water, KHSO₄ (5%) and finally withwater. Drying (Na₂SO₄) and concentration yielded the desired methylamide(100 mg, 96%) which exhibited the following data: ¹H NMR (CDCl₃, 300MHz) d 8.15 (br s, 1H), 7.63 (d, 1H), 7.42–7.18 (m, 18H), 6.81 (d, 1H),6.62 (d, 1H), 4.62 (m, 1H), 4.25 (m, 1H), 3.22 (dd, 1H), 3.18 (dd, 1H),2.92 (m, 2H), 2.64 (d, 3H), 2.58 (dd, 1H), 2.42 (dd, 1H), 1.63 (s, 9H),1.41 (s, 9H); ¹³C NMR (CDCl₃, 75 MHz) d 171.3, 171.1, 170.3, 169.2,149.9, 144.3, 135.8, 130.6, 129.9, 128.6, 128.3, 127.5, 125.1, 124.9,123.2, 119.6, 115.7, 84.1, 82.5, 68.3, 53.9, 50.2, 36.6, 36.0, 28.6,28.4, 27.5, 26.9; MS(Pos. FAB) 805 (MH+). The methylamide wasdeprotected to provide the title compound (72%); MS (Pos. FAB) 407(MH+), 429 (MNa+).

EXAMPLE 116N-[1-(1-Carboxy-2-hydroxy-ethylcarbamoyl)-2-(1H-indol-3-yl)-ethyl]-3-(2-mercapto-3-phenyl-propionylamino)-succinamicacid

The title compound was prepared by condensing H-Ser(But)OBut with theprotected dipeptide of Example 88, followed by deprotection andpurification by HPLC (40%): (Pos. FAB) ? (MH+).

EXAMPLE 117N-[2-(1H-indol-3-yl)-methoxycarbonyl-etjyl]-3-(2-mercapto-acetylamino)-succinamic acid

Esterification of the compound of Example 86 with methanol using EDCI,and in the presence of 10 mol % of DMAP gave, following deprotection,the title compound (41%): (Pos. FAB) 408 (MH+), 430 (MNa+).

EXAMPLE 118N-[2-(1H-indol-3-yl)1-ethyl]-3-(2-mercapto-3-phenyl-propionylamino)-succinamicacid

This compound was prepared by condensing tryptamine with2-(3-phenyl-2-tritylsulfanyl-propionylamino)-succinic acid 4-tert-butylester using the same coupling conditions as previously described.Deprotection and purification then provided the title compound (39%):(Pos FAB) 440 (MH+).

EXAMPLE 1192-(2-tert-Butoxycarbonylmethyl-5-tritylsulfanyl-pentanoylamino)-succinicacid di-tert-butyl ester

To H-Asp (O^(t)Bu)-O^(t)Bu hydrochloride salt (28 mg, 0.10 mmol) in dryDMF (2 mL) was sequentially added at room temperature2-(3-tritylsulfanyl-propyl)-succinic acid 4-tert-butyl ester (50 mg,0.10 mmol), HOBt (14 mg, 0.10 mmol), PYBOP (56 mg, 0.11 mmol) and DIPEA(37 μL, 0.20 mmol). The usual aqueous work up yielded the title compound(62 mg, 86%) as a colorless residue: ¹H NMR (CDCl₃, 400 MHz) δ 7.41–7.38(m, 6H), 7.29–7.25 (m, 6H), 6.60–6.57 (m, 1H), 4.68–4.56 (m, 1H),3.18–3.14 (m, 2H), 2.89–2.80 (m, 1H), 2.74–2.52 (m, 2H), 2.43–2.36 (m,1H), 2.32–2.19 (m, 1H), 2.12–2.07 (m, 2H), 1.83–1.80 (m, 2H), 1.60–1.53(m, 1H), 1.45–1.40 (m, 27H); ¹³C (DEPT) (CDCl₃, 100 MHz) δ 129.4, 127.7,126.4, 49.0, 48.7, 46.1, 42.5, 37.8, 37.6, 37.2, 31.6, 31.5, 29.6, 27.9,27.8, 27.7, 26.3, 26.2, 26.0.

EXAMPLE 120 2-(2-Carboxymethyl-5-mercapto-pentanoylamino)succinic acid

To the protected thiolate of Example 119 in CH₂Cl₂ (1 mL) was added at0° C. a solution of 60% TFA in CH₂Cl₂, containing 2% of ethanedithiol.Triisopropyl silane (1%) was then added and the reaction mixture wasstirred at room temperature for 1 hour after which it was firstconcentrated using a stream of argon and then under vacuum. The obtainedwhite solid was washed several times with a mixture of diethylether/hexanes (3:1). The obtained white solid was purified bysemi-preparative HPLC to provide the desired compound.

EXAMPLE 1213-(1-tert-Butoxycarbonyl-2-phenyl-ethylcarbamoyl)-6-tritylsulfanyl-hexanoicacid tert-butyl ester

The same peptide coupling protocol as described for Example 119 was usedand provided the title compound (70 mg, 100%): ¹H NMR (CDCl₃, 400 MHz) δ7.40–7.39 (m, 5H), 7.31–7.12 (m, 15H), 6.15 (d, 1H), 4.74–4.68 (m, 1H),3.20–3.15 (m, 2H), 3.11–2.98 (m, 2H), 2.55–2.49 (dd, 1H), 2.44–2.29 (m,1H), 2.24–2.16 (m, 1H), 2.12–2.05 (m, 2H), 1.84–1.81 (m, 2H),1.35 (4s,18H); ¹³C (DEPT) (CDCl₃, 400 MHz) δ 129.5, 129.4, 129.3, 128.2, 127.7,126.7, 126.4, 53.4, 53.3, 46.2, 46.1, 42.3, 42.2, 38.2, 38.1, 37.7,37.5, 31.5, 31.4, 31.3, 29.6, 27.9, 27.8, 26.3, 26.2, 26.0, 25.9.

EXAMPLE 122 2-(2-Carboxymethyl-5-mercapto-pentanoylamino)succinic acid

The deprotection leading to the final product was the same as that ofExample 120. Purification by semi-preparative HPLC gave the titlecompound as a white powder.

EXAMPLE 123 3-(Biphenyl-4-yl-ethylcarbamoyl)-2-phenethyl-pentanedioicacid

To an ice cooled solution of TFA (30%) in DCM (5 mL) containing water(5%) was added the compound of Example 55. The reaction mixture wasstirred for 2 h at room temperature, after which time, the solvent wasremoved. The residue was triturated with ether and evaporated. Theresidue was partitioned between sodium hydroxide (1N) and ether.Following separation, the alkaline phase was acidified to pH 1 using HCl(1N) and extracted with ethyl acetate. The recombined organic layer wasdried over sodium sulfate, filtered and evaporated. FAB: 460.1(MH⁺),FAB(M−H⁺): 458.0.

EXAMPLE 124N-[1-tert-Butoxycarbonyl-2-(1H-indol-3-yl)-ethyl]-3-(methoxycarbonylmethyl-amino)-succinamic acid tert-butyl ester

To a suspension of the dipeptide of Example 57 (100 mg, 0.23 mmol) indry THF (2 mL) was added DIPEA (40 μL, 0.46 mmol). After the mixturebecame a homogenous solution, it was cooled to 0° C. and methylbromoacetate (22 μL, 0.23 mmol) was introduced as a solution in dry THF(0.5 mL). The light suspension, which appeared shortly after, wasstirred at 0° C. for 10 min and then at room temperature for 5 hours.The mixture was then concentrated in vacuum, dissolved in ethyl acetateand washed with H₂O and brine. Drying (Na₂SO₄) and evaporation gave thetitle compound (92 mg) as a yellow foam. Flash chromatography (CH₂Cl₂ to5% methanol in CH₂Cl₂) gave the desired product in 62% yield: ¹HNMR(CDCl₃, 300 MHz) □ 8.52 (br s, 1H), 7.82 (d, 1H), 7.59 (d, 1H),7.38–7.02 (m, 4H), 4.75 (m, 1H), 4.18 (m, 1H), 3.68 (s, 3H), 3.61–3.21(m and dd, 3H), 3.14 (dd, 1H), 2.85 (m, 2H), 1.42 (s, 9H), 1.36 (s, 9H);MS (Pos. FAB) 504.5 (MH)⁺.

EXAMPLE 125 3-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-trityloxycarbamoylbutyric acid tert-butyl ester

Standard coupling between the building block of Example 63 and2-biphenyl-4-yl-ethylamine gave the crude product. Silica gelchromatography of the crude using 20–40% ethyl acetate/n-hexanesafforded the desired product in 50% yield as a white solid.

¹H-NMR(CDCl₃, 400 MHz)

1.39(s, 9), 1.87–2.48(m, 4H), 2.76–2.87(m, 3H), 3.45(m, 2H), 6.32(d,1H), 7.32(m, 15H), 7.43(m, 5H), 7.52–7.59(m, 4H), 7.82(d, 1H). FAB:668.9(MH⁺).

EXAMPLE 1263-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid

Deprotection of the compound of Example 125 by standard procedures,followed by HPLC purification, gave the title compound. ¹H-NMR (DMSO-d₆,400 MHz): 1.98 (dd, 1H), 2.21(m, 2H), 2.44 (dd, 1H), 2.71 (t, 2H), 2.93(m, 1H), 3.24 (dd, 2H), 7.31 (m, 3H), 7.44 (t, 2H), 7.57 (d, 2H), 7.62(d, 2H), 8.77 (s, 1H), 8.80 (t, 1H), 10.42 (s, 1H); ¹³C-NMR (DMSO-d₆,400 MHz, DEPT): 34.71, 35.4 29.3, 37.9, 40.2, 126.4, 127.2, 126.5,128.9; FAB: 371(MH⁺).

The compounds of Examples 127, 128–149 and 150 have all been preparedusing the same methods as described for the compounds of Examples 125and 126.

EXAMPLE 1273-[2-(1H-Indol-3-yl)-ethylcarbamoyl]-4-trityloxycarbamoyl-butyric acidtert-butyl ester

This compound was prepared by reacting the compound of Example of 63with tyramine. ¹H NMR (CDCl₃, 400 MHz):. 1.37(s, 9H), 1.39(s, 9H),1.89–2.37(m, 4H), 2.84(m, 1H), 3.20(m, 2H), 4.69(m, 1H), 6.77(m, 1H),7.23 (m, 21H), 8.25 (m, 1H); FAB: 731(MH⁺).

EXAMPLE 1284-Hydroxycarbamoyl-3-[2-(1H-indol-3-yl)-ethylcarbamoyl]-butyric acid

This compound was prepared by deprotection of the compound of Example127.

EXAMPLE 1293-[2-(4′-Cyano-biphenyl-4-yl)-ethylcarbamoyl]-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 withthe compound of Example 36.

EXAMPLE 1303-[2-(4′-Cyano-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid

This compound was prepared by deprotection of the compound of Example129.

EXAMPLE 1313-[2-(4-Pyridin-2-yl-phenyl)-ethylcarbamoyl]-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 withthe compound of Example 37.

EXAMPLE 1324-Hydroxycarbamoyl-3-[2-(4-pyridin-2-yl-phenyl)-ethylcarbamoyl]-butyricacid

This compound was prepared by deprotection of the compound of Example131.

EXAMPLE 133 3-(4-Phenyl-butylcarbamoyl)-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 with4-phenyl butylamine.

EXAMPLE 134 4-Hydroxycarbamoyl-3-(4-phenyl-butylcarbamoyl)-butyric acid

This compound was prepared by deprotection of the compound of Example133.

EXAMPLE 1353-(2-Naphthalen-1-yl-ethylcarbamoyl)-4-trityloxy-carbamoyl-butyric acidtert-butyl ester

This compound was prepared by reacting the compound of Example 63 withthe compound of Example 43.

EXAMPLE 1364-Hydroxycarbamoyl-3-(2-naphthalen-1-yl-ethylcarbamoyl)-butyric acid

This compound was prepared by deprotection of the compound of Example135.

EXAMPLE 137 3-(2-Phenoxy-ethylcarbamoyl)-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 with2-phenoxy ethylamine.

EXAMPLE 138 4-Hydroxycarbamoyl-3-(2-phenoxy-ethylcarbamoyl)-butyric acid

This compound was prepared by deprotection of the compound of Example137.

EXAMPLE 1393-[2-(4′-Hydroxy-biphenyl-4-yl)-ethylcarbamoyl]-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound Example of 63 withthe compound of Example 38.

EXAMPLE 1403-[2-(4′-Hydroxy-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid

This compound was prepared by deprotection of the compound of Example139.

EXAMPLE 141 3-(2,2-Diphenyl-ethylcarbamoyl)-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 withthe compound of Example 46.

EXAMPLE 142 3-(2,2-Diphenyl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyricacid

This compound was prepared by deprotection of the compound of Example141.

EXAMPLE 1433-[2-(4′-Dimethylamino-biphenyl-4-yl)-ethylcarbamoyl]-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 withthe compound of Example 39.

EXAMPLE 1443-[2-(4′-Dimethylamino-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid

This compound was prepared by deprotection of the compound of Example143.

EXAMPLE 1453-[2-(3′,4′-Dimethoxy-biphenyl-4-yl)-ethylcarbamoyl]-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 withthe compound of Example 35.

EXAMPLE 1464-Hydroxycarbamoyl-3-[2-(3′,4′-dimethoxy-biphenyl-4-yl)-ethylcarbamoyl]-butyricacid

This compound was prepared by deprotection of the compound of Example145.

EXAMPLE 147 3-(5-Hydroxy-pentylcarbamoyl)-4-trityloxycarbamoyl-butyricacid tert-butyl ester

This compound was prepared by reacting the compound of Example 63 with5-amino-pentan-1-ol.

EXAMPLE 148 4-Hydroxycarbamoyl-3-(5-hydroxy-pentylcarbamoyl)-butyricacid

This compound was prepared by deprotection of the compound of Example147.

EXAMPLE 1493-[(Biphenyl-4-ylmethyl)-carbamoyl]-4-trityloxycarbamoyl-butyric acidtert-butyl ester

This compound was prepared by reacting the compound of Example 63 withbiphenylmethylamine.

EXAMPLE 1503-[(Biphenyl-4-ylmethyl)-carbamoyl]-4-hydroxycarbamoyl-butyric acid

This compound was prepared by deprotection of the compound of Example149.

EXAMPLE 1513-(2-Biphenyl-4-yl-ethylcarbamoyl)-5-trityloxycarbamoyl-pentanoic acidtert-butyl ester

To a solution of the compound of Example 66 (300 mg, 0.6 mmol) inanhydrous acetonitrile (3 mL) were successively added DIEA (528 μL, 3mmol), 2-(4-biphenyl) ethylamine hydrochloride (280 mg, 1.2 mmol), andTBTU (234 mg, 0.72 mmol). The reaction mixture was stirred under argonfor 24 h. The mixture was partitioned between brine and ethyl acetate.The aqueous phase was extracted several times with ethyl acetate. Therecombined organic layer was extracted with HCl (1N, excess), brine, asaturated sodium bicarbonate solution, and brine followed by drying oversodium sulfate, filtration, and evaporation. The residue was flashedwith 40% ethyl acetate/hexanes to give the product as a white solid. ¹HNMR (CDCl₃, 400 MH

: 1.43(s, 9H); 1.57–1.81(m, 4H); 2.06(m, 1H); 2.36(m, 1H); 2.54(m, 1H);2,82(m, 2H); 3.48(m, 2H); 6.16(d, J=32.1 Hz, 1H); 7.34(m, 15H);7.42–7.60(m, 9H); 7.76(d, J=23.8 Hz, 1H).

EXAMPLE 1523-(2-Biphenyl-4-yl-ethylcarbamoyl)-5-hydroxycarbamoyl-pentanoic acid

This compound was prepared by deprotection of the compound of Example151, as previously described for the compound of Example 128.

EXAMPLE 1533-(Biphenyl-4-yl-ethylcarbamoyl)-6-phenyl-4-trityloxycarbamoyl-hexanoicacid tert-butyl ester

WSC.HCl (683 mg, 3.49 mmol) was dissolved in water (1 mL) and the pH wasadjusted to 4.8. The compound of Example 55 (300 mg, 0.582 mmol) wasdissolved in the same amount of THF and added to the former solution.O-Trityl hydroxylamine (505 mg, 1.746 mmol) was added and the mixturewas stirred for 24 h. The reaction mixture was partitioned between HCl(0.5N, excess) and ethyl acetate. The water phase was extracted severaltimes with ethyl acetate. The recombined organic layer was washed withbrine, a saturated sodium hydrogen carbonate solution, and brine. Thesolvent was evaporated, following drying over sodium sulfate, and thecrude was flashed with 30% ethyl acetate in hexanes. ¹H NMR(CDCl₃, 400MH

: 1.32(s, 9H); 1.50(m, 1H); 1.74(m, 1H); 2.32(m, 6H); 2.53(m, 2H);3.46(m, 2H); 7.00(m, 1H); 7.16–7.57(m, 30H); FAB: 774(MH⁺).

EXAMPLE 1543-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-hydroxycarbamoyl-6-phenyl-hexanoicacid

This compound was prepared by deprotection of the compound of Example153.

EXAMPLE 1554-Benzyloxycarbamoyl-3-(2-biphenyl-4-yl-ethylcarbamoly)-butyric acidtert-butyl ester

A solution of the compound of Example 72 (0.90 g, 2.19 mmol) inanhydrous DMF (3 mL) was treated successively with DIEA (1.9 mL),O-benzyl hydroxylamine hydrochloride (706 mg, 4.38 mmol), and TBTU (1.02g, 3.07 mmol). The reaction mixture was stirred for 24 h at roomtemperature. HCl (1M, excess) was added and the mixture was extractedseveral times with ethyl acetate. The recombined organic layer waswashed with brine, dried over sodium sulfate, filtered and evaporated.The residue was flashed with 30% ethyl acetate in hexanes and thedesired product was obtained as a white solid. ¹H NMR(CDCl₃, 400 MH

: 1.42(s, 9H); 2.33(m, 2H); 2.45(m, 1H); 2.63(m, 1H); 2.82(m, 2H);3.03(m, 1H); 3.47(m, 2H); 4.87(m, 2H); 6.02(s, broad, 1H); 7.25(m, 2H);7.34–7.45(m, 8H); 7.56(m, 4H).

EXAMPLE 1563-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid

A solution of TFA in DCM (30%, 5 mL) containing water (5%) was prepared.The solution was cooled in an ice bath and then the compound of theExample 155 (200 mg, 0.387 mmol) was added. The reaction mixture wasfirst stirred for 5 minutes at ° C., followed by 1 h at roomtemperature. The solvent was evaporated and the residue was trituratedseveral times with ether and then evaporated. The residue was dissolvedin wet methanol and hydrogenated using 10% Pd on charcoal. After 24 h,the reaction mixture was filtered over a celite pad and washedrepeatedly with methanol. The filtrate was evaporated and then subjectedto HPLC-purification.

EXAMPLE 1573-(2-Biphenyl-4-yl-ethylcarbamoyl)-5-(bis-benzyloxy-phosphoryl)-pentanoicacid tert-butyl ester

A solution of the compound of Example 77 (770 mg, 1.67 mmol) inanhydrous DMF (2 mL) was treated with DIEA (1.5 mL), 4-biphenylethylamine (672 mg, 3.34 mmol), and TBTU (775 mg, 2.34 mmol). The reactionmixture was stirred for 24 h at room temperature after which HCl (1M,excess) was added. The mixture was extracted several times with ethylacetate. The recombined organic layer was washed with brine, dried oversodium sulfate, filtered and evaporated. The residue was flashed with50% ethyl acetate in hexanes and the desired product was obtained as awhite solid. ¹H NMR (CDCl₃, 400 MH

: 1.41(s, 9H); 1.61–1.87(m, 4H); 2.15, 2.59(2dd, J₁=16.4 Hz. J₂=4.2 Hz,2H); 2.67(m, 1H); 2.80(m, 2H); 3.49(m, 2H); 4.96(m, 4H); 6.35(t, J=5.6Hz); 7.32(m, 13H); 7.42(m, 2H); 7.55(m, 4H); ³¹P NMR(CDCl₃, 400 MHz):33.05.

EXAMPLE 158 3-(2-Biphenyl-4-yl-ethylcarbamoyl)-5-phosphono-pentanoicacid

This compound was prepared by deprotection of the compound of Example157 using a procedure similar to that of the synthesis of the compoundof Example 156.

EXAMPLE 159N-[1-benzyloxycarbonyl-2-(1H-indol-3-yl)-ethyl]-3-[2-(diethoxy-phosphoryl)-4-phenyl-butyrylamino]-succinamicacid benzyl ester

To a stirred solution of the peptide of Example 83 (536 mg, 1.00 mmol)in DMF (3 mL) was sequentially added phosphonoacetic acid 79 (1.00mmol), HOBt (135 mg, 1.00 mmol) and PYBOP (520 mg, 1.00 mmol) at roomtemperature. DIPEA (523 μL, 3.00 mmol) was then added and the mixturewas stirred at room temperature for 18 h. The reaction mixture waspoured onto an aqueous solution of NaHCO₃ (5%) and extracted severaltimes with ethyl acetate. The combined organic extracts were washed withNaHCO₃ (5%), H₂O, KHSO₄ (5%) and H₂O. Drying (Na₂SO₄) and evaporationgave the crude product (780 mg) as a light oil. Flash chromatography(SiO₂, ethyl acetate:hexane (2:1) to 100% ethyl acetate) gave aseparable pair of diastereoisomers in a combined yield of 71%. The lesspolar diastereoisomer had the following spectroscopic data: ¹H NMR(CDCl₃, 400 MHz) □ 8.03 (br d, 2H), 7.61 (br d, 1H), 7.40–7.11 (m, 19H),6.82 (d, 1H), 5.15–4.82 (m, 5H), 4.87 (dd, 1H), 4.01 (m, 4H), 3.31 (d,2H), 3.08 (dd, 1H), 2.78–2.43 (m, 4H), 2.35 (m, 1H), 2.00 (m, 1H), 1.26(dt, 6H); ³¹P NMR (CDCl₃) δ 25.1; MS (Pos. FAB) 780.8 (M⁺).

The more polar diastereoisomer had the following spectroscopic data: ¹HNMR (CDCl₃, 400 MHz) □ 8.00 (br s, 1H), 7.58 (d, 1H), 7.39–7.17 (m,19H), 6.84 (d, 1H), 5.15–4.98 (s and dd, 4H), 4.91 (m, 2H), 4.03 (m,4H), 3.30 (d, 2H), 3.12 (dd, 1H), 2.65 (dd, 1H), 2.52 (m, 2H), 2.32–1.92(m, 2H), 1.78 (br s, 1H), 1.25 (q, 6H); ³¹P NMR (CDCl₃) δ 24.6; MS (Pos.FAB) 780.8 (M⁺).

EXAMPLE 160N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-(4-phenyl-2-phosphono-butyrylamino]-succinamicacid

The more polar peptide phosphonate of Example 159 (54 mg, 69 μmol) wasdissolved in dry CH₂Cl₂ (2 mL) followed by the addition of TMSBr (73 μL,552 μmol). The resulting pale yellow solution was stirred for 40 h. Thevolatile materials were evaporated in vacuum at room temperature and theobtained residue was suspended in a water/methanol mixture (20:1) andvigorously stirred for 2 h after which it was lyophilized to give thecrude phosphonic acid intermediate. This crude product was taken up inmethanol (5 mL) and treated with Pd/C powder (10%, 25 mg). The mixturewas stirred under an H₂ atmosphere (45 PSI) for 12 h and then thecatalyst was removed by filtration through a celite pad. The resultingmixture was concentrated to give the crude title compound which waspurified by HPLC.

EXAMPLE 161N-[1-benzyloxycarbonyl-2-(1H-indol-3-yl)-ethyl]-3-[1-(dimethoxy-phosphoryl)-3-phenyl-propylamino]-succinamicacid benzyl ester

To a stirred solution of H-AspTrp(OBn)-Trp(OBn) (536 mg, 1.00 mmol) inCH₂Cl₂ (4 ml) under argon atmosphere, was added DIPEA (175 μL, 1.00mmol) followed by the addition of hydrocinnamaldehyde (158 μL, 1.2 mmol)and Na₂SO₄ (0.71 g, 5.00 mmol). The heterogeneous mixture was stirred atroom temperature for 6 h and was filtered into another dry flask withthe Na₂SO₄ cake being washed with CH₂Cl₂ (1 ml). The solution wasmaintained under argon while being cooled to 0° C. To this stirringsolution was added trimethyl phosphate (208 μL, 1.76 mmol), followed byBF₃.Et₂O (150 μL, 1.20 mmol). The solution was allowed to warm slowly toambient temperature and was further stirred for an additional 15 h. Thereaction mixture was then diluted with CH₂Cl₂ and washed with ice-coldwater. The organic extracts were dried (Na₂SO₄) and then concentrated togive 700 mg of a yellow foam. Purification by flash chromatography usinga gradient of EtOAc in hexane (50% to 100%) provided a 1:1 mixture ofdiastereoisomers (0.31 g, 43%): ¹H NMR (CDCl₃) δ 8.10 (s, 1H), 8.06 (d,1H), 7.61 (d, 1H), 7.38–7.05 (m, 18H), 6.99 (d, 1H), 5.18–5.03(dd and s,4H), 4.90 (dd, 1H), 3.68 (m, 1H), 3.63 (d, 3H), 3.55 (d, 3H), 3.33 (m,2H) 2.90 (m, 1H), 2.75–2.52 (m, 4H), 2.01 (m, 2H), 1.75 (m, 2H); ³¹P NMR(CDCl₃) δ 29.6; MS (Pos. FAB) 725.4 (M⁺).

EXAMPLE 162N-[1-carboxy-2-(1H-indol-3-yl)-ethyl]-3-(3-phenyl-1-phosphono-propylamino)-succinicacid

This compound was deprotected by catalytic hydrogenation using the sameprotocol as presented for the compound of Example 160. MS(Pos. FAB):540(MNa⁺).

EXAMPLE 163N-[1-Benzyloxycarbonyl-2-(1H-indol-3-yl)-ethyl]-3-{[benzyloxy-(4-phenyl-butyl)-phosphinoyl]-amino}-succinamicacid benzyl ester

To a stirred solution of the phosphinate of Example 81 (144 mg, 0.50mmol) in dry degassed acetonitrile (1.5 mL), was addedbis-(trimethylsilyl)acetamide (73 μL, 0.30 mmol) followed immediately bythe addition of a solution of H-Asp(OBn)-Trp(OBn), [Example 83 (322 mg,0.60 mmol)] and Et₃N (209 μL, 1.5 mmol) in CCl₄/CH₃CN (3:1, 2 mL). Thereaction mixture was stirred at room temperature for 14 h and thenconcentrated in vacuum. Purification by flash chromatography using agradient of EtOAc in hexane (50% to 100%) provided the title compound(0.175 g, 44%) as a white residue: R_(f) (100% EtOAc)=0.5; ¹H NMR(CDCl₃) δ 8.5 (d, 1H), 7.6 (d, 1H), 7.5–6.9 (m, 25), 5.2–4.9 (m, 6H),4.83 (dd, 1H), 4.63 (dd, 1H), 4.22 (m, 1H), 3.91(d, 1H), 3.36 (dt, 1H),3.28 (dt,1H), 3.01 (dd, 1H), 2.65 (dt, 1H), 2.50 (m, 2H), 1.71–1.25 (m,6H); ³¹P NMR (CDCl₃) δ 35.6.

EXAMPLE 164N-[1-Carboxy-2-(1H-indol-3-yl)-ethyl]-3-{[hydroxy-(4-phenyl-butyl)-phosphinoyl]-amino}-succinamicacid

This compound was prepared using catalytic hydrogenation, following thesame protocol as presented above for the compound of Example 160.MS(Pos. FAB): 538(MNa⁺).

EXAMPLE 165 (5-tert-Butoxycarbonylmethyl-2,2-dimethyl-4,6-dioxo-[1,3]dioxan-5-yl)-acetic acid tert-butyl ester

To a solution of 2,2-dimethyl-1,3-dioxane-4,6-dione (5 g, 33 mmol) inchloroform (100 mL) was added potassium carbonate (27.6 g, 120 mmol) andbenzyltriethylammoniumchloride (16.6 g, 120 mmol). The reaction mixturewas stirred for 15 minutes and then treated drop wise with a solution oftert-butyl bromoacetate (18 mL, 120 mmol) in chloroform (50 mL). Theresultant mixture was stirred for 5 h at 50° C. After cooling to roomtemperature, water was added (100 mL). After shaking, the organic phasewas separated and the water phase was extracted two times withchloroform. The recombined organic layer was washed with brine, driedover sodium sulfate, filtered and evaporated. The resultant light yellowsolid was dissolved in ether (50 mL) and washed several times with waterfollowed by drying over sodium sulfate, filtration, and evaporation.Most of the solvent was evaporated and the residue was triturated withhexanes (20 mL). After about 30 minutes standing at room temperature,crystallization of the desired product began. The mixture wasrefrigerated. After 24 h, the product was filtered and washed with icecold ether followed by drying under high vacuum. ¹H NMR(CDCl₃, 300 MHz):1.39(s, 18H); 1.90(s, 6H); 2.95(s, 4H).

EXAMPLE 166 3,3-Dicarboxy-pentanedioic acid di-tert-butyl ester

The compound of Example 165 (200 mg, 0.54 mmol) was dissolved in amixture of THF(10 mL) and water (2 mL). Lithium hydroxide monohydrate(46 mg, 1.07 mmol) was added and the mixture was stirred for 24 h atroom temperature. The solvent was evaporated and the residue wasdissolved in water (5 mL) and extracted with ether. The water phase wasacidified by HCl to pH1 and extracted with ethyl acetate. After dryingover sodium sulfate, filtration and evaporation, the solvent wasevaporated to dryness. ¹H NMR(CDCl₃, 300 MHz): 1.43(s, 18H); 3.10(s,4H).

EXAMPLE 167 3-Carboxy-pentanedioic acid di-tert-butyl ester

The compound of Example 166 (170 mg, 0.512 mmol) was dissolved in ethylacetate. Formic acid (61 μL, 1.53 mmol) was added and the mixture washeated at 80° C. for 3 h. After cooling to room temperature, thereaction mixture was extracted three times with water, dried (sodiumsulfate), filtered and evaporated to dryness. ¹H NMR(CDCl₃, 300 MHz):1.43(s, 18H); 2.50, 2.65(2dd, J₁=16.7 Hz, J₂=6.2 Hz, 4H); 3.20(p, J=6.2Hz, 1H).

EXAMPLE 168 3-(2-Naphthalen-2-yl-ethylcarbamoyl)-pentanedioic aciddi-tert-butyl ester

A solution of the compound of Example 166 (144 mg, 0.5 mmol) inanhydrous DMF (2 mL) was treated successively with triethylamine (92 μL,0.65 mL), the compound of example 43 (140 mg, 0.6 mmol), DIC(96 μL, 0.6mmol), and HOBt (82 mg, 0.6 mmol). The reaction mixture was stirred for24 h at room temperature. HCl (1M, excess) was added and the mixture wasextracted several times with ethyl acetate. The recombined organic layerwas washed with brine, dried over sodium sulfate, filtered andevaporated. The residue was flashed with 20–25% ethyl acetate in hexanesand the desired product was obtained as a white solid. ¹H NMR(CDCl₃, 300MH

1.40(s, 18H); 2.31, 2.62(2dd, J₁=16.5 Hz, J₂=4.2 Hz, 4H); 2.96(m, 3H);3.59(m, 2H); 6.25(m, 1H); 7.34–7.49(m,3H); 7.69(s, 1H); 3.82(m, 3H).

EXAMPLE 169 3-(2-Naphthalen-2-yl-ethylcarbamoyl)-pentanedioic acid

Deprotection of the compound of example 168 by standard procedurefollowed by HPLC purification gave the title compound.

General Procedure to Determine PHEX Enzymatic Activity

The soluble form of the metallopeptidase PHEX (sPHEX) was purified tohomogeneity according to the methods of Boileau and Crine (WO 00/50580).sPHEX quantity was measured with the Bradford assay (BioRad) using BSAas a standard.

EXAMPLE 170 Determination of sPHEX Enzymatic Activity

sPHEX enzymatic activity was determined using an HPLC method (WO00/50580).

EXAMPLE 171 Determination of sPHEX Activity Using a FluorogenicSubstrate

sPHEX activity was determined using a fluorogenic substrate in which anincrease of fluorescence was proportional to substrate conversion toproducts.

Three fluorogenic substrates were proven to be particularly good sPHEXsubstrates: Abz-GFRDWK-Dnp (S1), Abz-DHLSDTSTQ-edDnp (S2) andAbz-GFSDYK-Dnp (S3). sPHEX showed the best kinetic parameters for S3that was therefore the commonly used substrate. S3 was synthesized atDr. Gilles Lajoie's laboratories (University of Western Ontario, Canada)by standard solid phase peptide synthesis. The fluorescent reactionproduct was excited at 320 nm to fluoresce at 420 nm. Fluorescence wasrecorded at 420 nm every five minutes over a one-hour period on afluorescence plate reader (Perkin-Elmer, HTS-7000) to calculate theinitial rate of the reaction. Typically, the reaction was carried out in200 μl of buffer M (50 mM Mes(NaOH) pH 6.5, 150 mM NaCl) containing 100ng of purified sPHEX and 20 μM of fluorigenic substrate to initiate thereaction. Enzymatic activity was determined by subtracting either theinitial rate of substrate in buffer (solvolysis), or the typicalreaction rate in which 5 mM EDTA was added prior to initiating thereaction.

sPHEX inhibitor IC50 determinations were done using the tested inhibitorat various concentrations coming from a serial dilution (usually rangingfrom 10⁻³ M to 10⁻¹² M) in the reaction described above. IC50 valueswere calculated using the iterative four parameters non-linearregression formula from the GraphPad™ software (Prism).

To establish the relationship between recorded fluorescence and molaryield of the enzymatic reaction, a standard curve of fluorescence versusthe concentration of the reaction fluorescent adduct was established bymeasuring the fluorescence of the Abz-GFS peptide at differentconcentration in buffer M.

The assessment of sPHEX biological activity using one of theabove-mentioned substrates, or another one, need not be limited to anenzymatic activity of sPHEX. In accordance with one embodiment, thebinding of sPHEX to at least one of its substrates, and in particularone of S1, S2 and S3 could be carried-out. Such determination of sPHEXbiological activity could provide the means to screen and identifymodulators of sPHEX biological activity (e.g. binding, enzymaticactivity and otherwise).

In accordance with such embodiment, there is provided a kit forscreening and identifying sPHEX modulation of sPHEX biological activitycomprising a sPHEX and a sPHEX substrate selected from the groupconsisting of S1, S2 and S3.

Abbreviations used: Abz: aminobenzoic acid; Dnp: dinitrophenyl; edDnp:ethylenediamine-Dnp; G: glycine; F: phenylalanine; R: arginine; D:aspartic acid; W: tryptophan; K: lysine; H: histidine; L: leucine; S:serine; T: threonine; Q: glutamine; Y: tyrosine; BSA: bovine serumalbumin; IC50: inhibitor concentration at which 50% of the enzyme isinhibited.

The following table (Table 1) lists a series of exemplary compounds asdescribed herein as well as their respective biological activity (IC₅₀).

TABLE 1 Exemplary in vitro activity. Identification IC₅₀ (Example No)Structure (μM) 87

0.600 89

0.011 90

0.068 91

0.094 92

0.005 93

0.048 94

0.004 95

0.007 96

0.004 97

0.003 98

0.008 99

0.008 100

0.006 101

0.018 102

0.020 103

0.022 104

0.015 106

0.006 107

0.170 108

0.007 109

0.007 110

0.038 111

0.090 112

0.015 114

0.320 115

1.3 116

0.004 117

2.6 118

0.140 126

0.017 130

0.190 132

0.240 134

0.560 138

0.860 140

0.360 142

0.054 144

5.0 148

1.7 150

0.270 152

1.3 162

9.7 169

19General Procedure to Monitor Protection of a PHEX Substrate by a PHEXInhibitor.

EXAMPLE 172 Inhibition of sPHEX

In general, in order to show inhibition of sPHEX, the enzyme isincubated in the absence or the presence of the inhibiting compound, anda substrate is added in conditions that are suitable for sPHEX enzymaticactivity. Hydrolysis of the substrate in the absence or the presence ofthe inhibiting compound is monitored.

For example, 1.0 μg of purified sPHEX was first incubated at 37° C. for15 min with or without 1×10⁻⁵M of compound MH-2-64C (FIG. 1) in 90 μl 50mM MES (2-(N-morpholino)ethanesulfonic acid) pH 6.5, 150 mM NaCl. Then,2.5 μg of peptide PTHrP₁₀₇₋₁₃₉ in 10 μl of the same buffer were addedand the incubation continued for 30 min. The reaction mixture alsocontained 1.3 μg of the amino acid Phe which was used as an internalstandard. After the incubation period, the reaction was stopped by theaddition of EDTA to a final concentration of 5 mM. Detection of cleavageproducts was performed by reverse phase high performance liquidchromatography (RP-HPLC) on a C18 μBondapak™ analytical column (Waters,Mississauga, ON, Canada) with a UV detector set at 220 nm. Peptides wereresolved with a linear gradient of 10% to 50% mobile phase B in 15 minat 40° C. with a flow rate of 1.0 ml/min [mobile phase A=0.1%trifluoroacetic acid; mobile phase B=80% acetonitrile (CH₃CN), 0.1%trifluoroacetic acid]. Results were quantified by comparing the areaunder the undigested peak of PTHrP₁₀₇₋₁₃₉, after normalization for theamount of Phe present in the sample.

In the absence of sPHEX, no digestion of PTHrP₁₀₇₋₁₃₉ (elution time 12.9min) was evident (FIG. 2A). In the presence of sPHEX, however,degradation of approximately 75% of the peptide was observed (FIG. 2B).(The peak eluting at 5.7 min corresponds to the Phe used as internalstandard). Digestion of PTHrP₁₀₇₋₁₃₉ by sPHEX resulted in the productionof four degradation products eluted at 9.1, 9.2, 12.1, and 12.5 min(FIG. 2B). In the presence of compound MH-2-64C (compound of Example126), a PHEX inhibitor, degradation of PTHrP₁₀₇₋₁₃₉ was fully prevented(FIG. 2C). It can be concluded that a PHEX inhibitor can protect a PHEXsubstrate from degradation and thus be useful in the identification ofsuch substrates.

General Procedure to Test the Effects of PHEX Inhibitor onMineralization by Cultured Cells.

EXAMPLE 173 Stimulation of Mineralization in Cultured Cells by PHEXInhibitors

In general, to show stimulation of mineralization in cultured cells byPHEX inhibitors, cells of osteoblast lineage are incubated in conditionsthat favors the mineralization process in the absence and the presenceof the PHEX inhibiting compound. This cellular model is used to studyosteoblasts proliferation and differentiation, and to follow themineralization process.

Mineralization can be monitored by standard techniques such as von Kossastaining or 45Ca incorporation in the extracellular matrix.

For example, cells were enzymatically isolated from the calvaria of21-day old Wistar rat fetuses by sequential digestion with collagenaseas described previously (Bellows et al., 1986). Cells obtained from thelast four of the five digestion steps were plated in T-75 flasks inα-MEM containing 15% FBS (Cansera) and antibiotics comprising 100 μg/mlpenicillin G (Sigma-Aldrich), 50 μg/ml gentamycin (Life Technologies),and 0.3 μg/ml fungizone (Life Technologies). After 24 h incubation,attached cells were washed with PBS to remove nonviable cells and otherdebris, and then collected by trypsinization using 0.2% trypsin incitrate saline. Cells were plated in 12-well plates at 9.0×10³cells/well in α-MEM containing 10% FBS and antibiotics. After 24 hincubation, the medium was changed and supplemented with 50 μg/mlascorbic acid, 10 mM sodium β-glycerophosphate and 1×10⁻⁸M dexamethasone(day 1). Cells were treated with varying concentrations of PHEXinhibitor or vehicle from day 1 to 25 of culture. Medium was changedevery 2–3 days. All dishes were incubated at 37° C. in a humidifiedatmosphere in 95% air 5% CO₂ incubator.

At day 18, 21 and 25 of rat calvaria osteoblasts culture, mineralizationwas determined by ⁴⁵Ca incorporation assay as described previously(Ecarot et Desbarats, 1999). Culture medium was replaced by fresh □-MEMmedium containing 10% FBS, 1 □Ci/ml ⁴⁵CaCl₂ (5–30 Ci/g Ca, ICN), andvarying concentrations of PHEX inhibitor or vehicle. Cells were labelledfor 5 h at 37° C. in a humidified atmosphere in 95% air 5% CO₂incubator. Labelling medium was then removed and cells were incubatedwith unlabeled culture medium for 15 min and rinsed three times with0.9% NaCl. Cell layers were solubilized in 12.5% trichloroacetic acidand aliquots counted for radioactivity.

When cultured in the conditions described above, fetal rat calvariacells show incorporation of ⁴⁵Ca in the extracellular matrix around day15 (FIG. 3). The addition of the PHEX inhibitor MH-2-64C (compound ofExample 126) at a concentration of 1×10⁻⁵M, a concentration that totallyinhibits PHEX, reduced significantly ⁴⁵Ca incorporation by the cells(FIG. 3), suggesting that the mineralization process has been negativelyaffected by this compound. This result is consistent with reports thatcultured osteoblasts from the Hyp mouse, that lack an active PHEXprotein (Beck et al. 1997), show impaired mineralization (Nesbitt et al.1999). Surprisingly, lower concentrations of PHEX inhibitorsignificantly stimulated the incorporation of ⁴⁵Ca by the cultured cells(FIG. 3). A biphasic effect of the inhibitor on the ⁴⁵Ca incorporationrate was therefore observed: at high dose (10⁻⁵ M) an inhibition ofmineralization was observed whereas a low dose (10⁻⁶ M) (Data not shown)of inhibitor increased the mineralization rate. These results suggestthat low doses of PHEX inhibitors could be used to stimulate themineralization process.

General Procedure to Show Stimulation of Bone Lesion Repair in thePresence of Low Doses of PHEX Inhibitor.

EXAMPLE 174 Osteogenic Properties of PHEX Inhibitors

Osteogenic properties of PHEX inhibitors can be shown in a model of ratmandibular defect. For example, a 2 mm size bone defect was created inthe rat mandible as described previously (Vu et al., 1999). Rats wereanesthetized and the vestibular surface of the right mandibular ramuswas exposed as follow: an incision was made through the skin along animaginary line forming a 90° angle with the lower lip to access themuscle layer. A scalpel blade was used to make an incision parallel tothe fibers of the deep portion of masseter muscle, about 2 mm posteriorto the facial artery (largest branch of the external carotid), in theportion where it gives rise to the labial mandibular artery. Theperiosteum was elevated and the underlying bony surface exposed. A 2mm×2 mm bony window was drilled between the second and third molar usinga slow-speed dental drill with a carbide round burr size 1.4 mm(Brasseler) and a drill size 2 mm (Straumann Canada Ltd). Salineirrigation was used during the drilling. Porous hydroxyapatite particles(Fin-Ceramica Faenza s.r.l.) with pore size ranged from 450 to 600 μmwere accommodated in the bony hole and pushed into it. The particles hadbeen incubated previously in solutions of varying concentrations of PHEXinhibitor or vehicle for 16 to 24 hours at 4° C. Once the bony windowwas filled the lesion was covered with a membrane of hydroxyapatite(Fin-Ceramica Faenza s.r.l.) which was fixed to the surrounding bonesurface with topical tissue adhesive. The muscle fibers were carefullyrepositioned on the mandible and sutured with absorbable surgicalsutures (Chromic Gut, Ethicon Inc.). The skin was sutured for totalcoverage with silk sutures (Sherwood Davis & Geck, Wayne). After 7 daysof treatment the bone lesion was exposed and varying concentrations ofPHEX inhibitor or vehicle was reapplied to hydroxyapatite particles.Treatment was continued for another 7 days.

After the 14 day healing period, bone formation at the defect wasevaluated by scanning electron microscopy and microcomputed tomography.Animals were sacrificed by intravascular perfusion through the leftventricle with 4% paraformaldehyde and 0.1% glutaraldehyde solution.Treated hemimandibles were taken, immersed in the fixative overnight at4° C. then washed and kept in 0.1 M cacodylate buffer pH 8.0 untilanalysis. Specimens for quantification by scanning electron microscopywere dehydrated in a graded alcohol series and embedded in LR Whiteresin (Marivac) for further sectioning along their longitudinal axis.Computerized images of the sections were acquired using a JEOL, JSM-6460LV scanning electron microscope operated in the backscattered mode at 20kV. Amount of bone tissue present in the sections was quantified bymanual tracing using the MOP-3 system (Carl Zeiss) and was expressed asa percentage of the total defect area.

Analysis of the defects treated with implant of porous hydroxyapapatitein absence of PHEX inhibitor show bone formation after 14 days ofhealing (FIG. 4). The addition of the PHEX inhibitor MH-2-64C (compoundof Example 126) to hydroxyapatite particles at a concentration of 1×10⁻⁵M, a concentration that totally inhibits PHEX, reduced significantlybone formation compared to saline (FIG. 4) suggesting that the rate ofhealing has been negatively affected by this compound. On the contrary,lower concentrations of PHEX inhibitor significantly stimulated boneformation (FIG. 4). These results are consistent with the observationthat PHEX inhibitors have a biphasic effect on mineralization bycultured cells, and also suggest that low doses of PHEX inhibitors couldbe used in vivo as osteogenic agents in order to stimulate the bonehealing process. These findings are supported by microcomputedtomography measurements made on one representative specimen from eachgroup (FIG. 5). 3D reconstruction pictures of defect implanted withhydroxyapatite particles in presence of a high or a low dose of PHEXinhibitor show more bone around particles soaked in low dose ofinhibitor than around particles soaked in saline. Again, a highinhibitor dose reduced bone formation. These observations strengthen theuse of a low dose of PHEX inhibitor as an osteogenic agent in medicalapplications.

General Procedure to Use PHEX Inhibitors as Therapeutics.

EXAMPLE 175 Effect of a PHEX Inhibitor of the Present Invention onCirculating Phosphate

The administration of a therapeutically effective amount of a PHEXinhibitor of the present invention induces a normalization ofcirculating phosphate, thus reducing, or preferably preventing,hyperphosphatemia or hyperparathyroidism and the appearance of theirconsequences. Table 2 below lists examples of causes ofhyperphosphatemia.

TABLE 2 Causes Of Hyperphosphatemia Binding to serum proteins Plasmacell dyscrasias Decreased renal excretion Renal insufficiencyHypoparathyroidism Pseudohypoparathyroidism, types I and II Tumoralcalcinosis Pseudoxanthoma elasticum Infantile hypophosphatasiaHyperostosis Hyperthyroidism Growth hormone activity Adrenalinsufficiency Bisphosphonate therapy Increased intestinal absorptionPhosphorus-containing cathartics Vitamin D ingestion Granulomatousdiseases producing vitamin D Sarcoidosis Tuberculosis Internalredistribution Acute metabolic acidosis Lactic acidosis Acuterespiratory acidosis Lactic acid infusion Reduced insulin levelClonidine administration Cellular release Rhabdomyolysis Organinfarction Tumor lysis Burkitt's lymphoma Lymphoblastic lymphomaMetastatic small cell carcinoma Thyrotoxicosis Acute hemolysisParenteral administration Intravenous phosphate salts Lipid(phospholipid) infusion Spurious hyperphosphatemia ThrombocytosisHyperlipidemia

The PHEX inhibitors are administered to rats weighing about 250 g at adose of 10 mg/kg. The control group consists of another group of ratswhere the same vehicle is administered but without the PHEX inhibitor.Serum and urine are obtained from the test animals using standardmethods. The phosphate concentration in both serum and urine is measuredby standard methods. PHEX inhibitors capable of inducing a change inphosphate concentration are said to be hypophosphatemic or phosphaturic.Such compounds are the preferred “hypophosphatemic or phosphaturic PHEXinhibitors” for the purpose of treating hyperphosphatemic patients orfor treating patients requiring management of their phosphate levelsthrough other means than normal physiological responses includinghyperparathyroidism.

The term “effective amount” is used herein to refer to an amountsufficient to induce significant improvement in the disease or conditionto be prevented or treated. Without being so limited, in specificembodiments, it refers to an amount in the range of 0.001 mg/Kg/day to100 mg/Kg/day.

The “least effective dose” is the minimum dose that is required toinduce a significant reduction in serum phosphate, PTH concentration orto normalize other known phosphate active agents including FGF23 (orother phosphatonins). Similarly, “the least significant dose” is theminimum dose required to maintain normal phosphate levels to prevent orreduce an increase in PTH or to normalize other known phosphate activeagents including FGF23 (or other phosphatonins). Preferably, the therapywill be initiated with a least effective dose.

The treatment preferably involves the administration of a “phosphaturicPHEX inhibitor” for a period of time sufficient to either achieve areduction in phosphate or PTH blood concentration, another knownphosphate active agent including FGF23 concentration or phosphatonins(hereinafter the “blood parameters”) or maintain the blood parameters ata normal level if the reduction was already successfully achieved.Preferably, when reduction of these parameters is sought, the netreduction is about 25% of the difference between the patient value andthat of the normal population or, more preferably, at least about 50% ofthe difference between the patient's value and that of the normalpopulation. The specific period of time sufficient to achieve thisreduction in the subject blood parameters may depend on a variety offactors. Such factors include, for example, the specific phosphaturicinhibitor employed, the amount administered, the age and gender of thesubject, the specific disorder to be treated, concomitant therapiesemployed (if any), the general physical health of the subject (includingthe presence of other disorders), the severity of the disease in theindividual, and the nutritional habits of the individual.

As used herein “administering” includes any method which, in soundmedical practice, delivers a hypophosphatemic inhibitor of the presentinvention to a patient in need thereof, in such a manner so as to beeffective in achieving a reduction in the blood parameters. Thehypophosphatemic PHEX enzyme inhibitor may be administered by any of avariety of known methods of administration, e.g., orally,dermatomucosally (for example, dermally, sublingually, intranasally, andrectally), parenterally (for example, by subcutaneous injection,intramuscular injection, intra-articular injection, intravenousinjection), and by inhalation. Thus, specific modes of administrationinclude, for example, oral, transdermal, mucosal, sublingual,intramuscular, intravenous, intraperitoneal, subcutaneousadministration, and topical application. A preferred mode for deliveringthe hypophosphatemic PHEX enzyme inhibitors is orally, for as long andas frequently as medically required. The period and frequency isadjusted after regular measurement of serum phosphate, PTH and vitamin Dmetabolites.

The present invention also relates to a method of treatinghyperphosphatemia Thus, a preferred method of this invention comprisingthe steps of performing a diagnostic on a human subject for thedetection of hyperphosphatemia, including its most frequentmanifestations, secondary hyperparathyroidism and renal osteodystrophyas well as other metabolic conditions requiring phosphate managementand, upon obtaining a positive result from the diagnostic, administeringa phosphaturic PHEX enzyme inhibitor according to the methods of thisinvention. Suitable diagnostics for the detection of hyperphosphatemia,including its most frequent manifestations, secondaryhyperparathyroidism and renal osteodystrophy, are well known in the art.Such methods include the measurement of the blood, serum, plasma orurinary phosphate or the measurement of the blood, serum or plasma PTHas well as other diagnostic chemistries related to phosphate metabolismincluding FGF23 and phosphatonins.

EXAMPLE 176 Dosage Forms

The PHEX enzyme inhibitors as described herein may be administered inany of a variety of pharmaceutically acceptable compositions. Suchcompositions comprise an active ingredient and a pharmaceuticallyacceptable carrier. Accordingly, compositions for administering the PHEXenzyme inhibitor comprise:

(a) from about 1.0 mg to about 1 000.0 mg of a PHEX enzyme inhibitor;and

(b) a pharmaceutically acceptable carrier.

Pharmaceutically-acceptable carriers include solid or liquid fillerdiluents or encapsulating substances, and mixtures thereof, that aresuitable for administration to a human or lower animal. The term“compatible”, as used herein, means that the components of thepharmaceutical composition are capable of being co-mingled with thehypophosphatemic PHEX enzyme inhibitor, and with each other, in a mannersuch that there is no interaction, which would substantially reduce thepharmaceutical efficacy of the pharmaceutical composition under ordinaryuse situations. Pharmaceutically acceptable carriers must, of course, beof sufficiently high purity and sufficiently low toxicity to render themsuitable for administration to the humans or lower animals beingtreated.

Some examples of the substances which can serve as pharmaceuticalcarriers are: sugars, such as lactose, glucose and sucrose; starches,such as corn starch and potato starch; cellulose and its derivatives,such-as sodium carboxymethylcellulose, ethylcellulose, celluloseacetate; powdered tragacanth; malt; gelatin; talc; stearic acid;magnesium stearate; vegetable oils, such as peanut oil, cottonseed oil,sesame oil, olive oil, corn oil and oil of theobroma; polyols such aspropylene glycol, glycerin, sorbitol, mannitol, and polyethylene glycol;agar; alginic acid; pyrogen-free water; isotonic saline; phosphatebuffer solutions; wetting agents and lubricants such as sodium laurylsulfate; coloring agents; flavoring agents; and preservatives. Othercompatible pharmaceutical additives and hypophosphatemic PHEX enzymeinhibitor may be included in the pharmaceutically acceptable carrier foruse in the compositions of the present invention.

The choice of a pharmaceutically-acceptable carrier to be used inconjunction with the active substance is determined by the way theactive substance is to be administered. If the active is to be injected,the preferred pharmaceutical carrier is sterile water, physiologicalsaline, or mixtures thereof. The pH of such parenteral composition ispreferably adjusted to about 7.4. Suitable pharmaceutically acceptablecarriers for topical, anal or vaginal applications include those knownin the art for use in creams, gels, tapes, patches, and similar topicaldelivery means. The active could also be administered intra-nasally orpulmonary with a suitable carrier as the conditions will require.

The pharmaceutically-acceptable carrier employed in conjunction with thephosphaturic PHEX enzyme inhibitor is used at a concentration sufficientto provide a practical size to dosage relationship. Thepharmaceutically-acceptable carriers, in total, may comprise from about0.1% to about 99.9% by weight of the pharmaceutical compositions of thepresent invention, preferably from about 5% to about 80%, and mostpreferably from about 10% to about 50%.

As indicated, the preferred method of administering a phosphaturic PHEXenzyme inhibitor of the present invention is dependent upon the class ofactive being administered. For the phosphaturic PHEX inhibitors, thepreferred method of administration is orally, in a unit-dosage form(i.e., a dosage form containing an amount of active suitable foradministration in one single dose, according to sound medical practice).

Preferred unit dosage forms include tablets, capsules, suspensions, andsolutions, comprising a safe and effective amount of active.Pharmaceutically-acceptable carriers suitable for the preparation ofunit dosage forms for oral administration are well known in the art.Their selection will depend on secondary considerations like taste,cost, shelf stability, which are not critical for the purposes of thepresent invention, and can be made without difficulty by a personskilled in the art. Preferably, oral unit dosage forms of the PHEXenzyme inhibitor comprise from about 1.0 mg to about 1000 mg of theinhibitor.

Although the present invention has been described hereinabove by way ofpreferred embodiments thereof, it can be modified without departing fromthe spirit and nature of the subject invention as defined in theappended claims.

REFERENCES

The following references, to the extent that they provide exemplaryprocedural or other details supplementary to those set forth herein, arespecifically incorporated herein by reference.

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1. A compound of Formula (I), or a pharmaceutically acceptable saltthereof:

wherein A is a zinc ligand or zinc ligand bearing moiety selected fromthe group consisting of:

B is absent: R is hydrogen or lower alkyl; R₁ is hydrogen or loweralkyl; R₂ is hydrogen, or lower alkyl; R₃ is hydrogen or lower alkyl; R₄is lower alkyl, substituted lower alkyl, cycloalkyl-(CH₂)_(w)—,aryl-(CH₂)_(w)—, substituted aryl —(CH₂)_(w)— or heteroaryl-(CH₂)_(w)—;R₅ is hydrogen, lower alkyl, substituted lower alkyl,cycloalkyl-(CH₂)_(x)—, aryl-(CH₂)_(x)—, substituted aryl-(CH₂)_(x)—, orheteroaryl-(CH₂)_(x)—; R₈ and R₉ are independently selected from thegroup consisting of hydrogen, alkyl, substituted alkyl, aryl-(CH₂)_(y)—,substituted aryl-(CH₂)_(y)—, heteroaryl-(CH₂)_(y)—,

R₁₀ is alkyl, substituted alkyl, cycloalkyl-(CH₂)_(y), aryl-(CH₂)_(y)—,substituted aryl-(CH₂)_(y)— or heteroaryl-(CH₂)_(y)—; R₁₃ is hydrogen,lower alkyl, cycloalkyl or phenyl; R₁₄ is hydrogen, lower alkyl, loweralkoxy or phenyl; R₁₅ is lower alkyl or aryl-(CH₂)_(y)—; D is —COOH, Eis hydrogen, —COOH, —CONH₂, —CONH(lower alkyl), —CON(lower alkyl)₂,—CONH—(CH₂)_(z)-aryl, —CON(—(CH₂)_(z)-aryl)₂, —CO-amino acid, —CH₂COOH,CH₂OH, —CH₂CH₂OH, or —COOR₁₆; R₁₆ is selected from the group consistingof hydrogen, alkyl, substituted alkyl, aryl-(CH₂)_(y)—, substitutedaryl-(CH₂)_(y)—, heteroaryl-(CH₂)_(y)—,

C is carbon; H is hydrogen; O is oxygen; N is nitrogen; S is sulfur; Pis phosphorus; v is zero; w is zero or an integer ranging from 1 to 4; xis an integer ranging from 1 to 4; y is zero or an integer ranging from1 to 6; and z is zero, one, two, or three.
 2. The compound of claim 1,wherein R₁ and R₃, when v=0, are connected together to form an alkylenebridge of 3 carbon atoms representing with the carbon atoms to whichthey are attached a cyclopentane ring.
 3. The compound of claim 1,wherein R₁ and R₃, when v=0, are connected together to form an alkylenebridge of 4 carbon atoms representing with the carbon atoms to whichthey are attached a cyclohexane ring.
 4. The compound of claim 1,wherein R and R₄ are connected together to form an alkylene bridge of 3carbon atoms representing with the nitrogen and carbon atoms to whichthey are attached a pyrrolidine ring.
 5. The compound of claim 1,wherein R and R₄ are connected together to form an alkylene bridge of 4carbon atoms representing with the nitrogen and carbon atoms to whichthey are attached a piperidine ring.
 6. The compound of claim 1, whereinthe compound is further defined as:3-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid;3-[2-(4′-Cyano-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid;4-Hydroxycarbamoyl-3-[2-(4-pyridin-2-yl-phenyl)-ethylcarbamoyl]-butyricacid; 4-Hydroxycarbamoyl-3-(4-phenyl-butylcarbamoyl)-butyric acid;4-Hydroxycarbamoyl-3-(2-phenoxy-ethylcarbamoyl)-butyric acid;3-[2-(4′-Hydroxy-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid; 3-(2,2-Diphenyl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid;3-[2-(4′-Dimethylamino-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid; 4-Hydroxycarbamoyl-3-(5-hydroxy-pentylcarbamoyl)-butyric acid;3-[(Biphenyl-4-ylmethyl)-carbamoyl]-4-hydroxycarbamoyl-butyric acid;N-[1-carboxy-2-(1H-indol-3-yl)-ethyl]-3-(3-phenyl-1-phosphono-propylamino)-succinicacid; or 3-(2-Naphthalen-2-yl-ethylcarbamoyl)-pentanedioic acid.
 7. Thecompound of claim 6, wherein the compound is3-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid.
 8. Apharmaceutical composition comprising a therapeutically effective amountof a compound of claim 1 and a physiologically acceptable carrier orexcipient.
 9. The pharmaceutical composition of claim 8, wherein thecompound is further defined as:3-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid;3-[2-(4′-Cyano-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid;4-Hydroxycarbamoyl-3-[2-(4-pyridin-2-yl-phenyl)-ethylcarbamoyl]-butyricacid; 4-Hydroxycarbamoyl-3-(4-phenyl-butylcarbamoyl)-butyric acid;4-Hydroxycarbamoyl-3-(2-phenoxy-ethylcarbamoyl)-butyric acid;3-[2-(4′-Hydroxy-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid; 3-(2,2-Diphenyl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid;3-[2-(4′-Dimethylamino-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid; 4-Hydroxycarbamoyl-3-(5-hydroxy-pentylcarbamoyl)-butyric acid;3-[(Biphenyl-4-ylmethyl)-carbamoyl]-4-hydroxycarbamoyl-butyric acid;N-[1-carboxy-2-(1H-indol-3-yl)-ethyl]-3-(3-phenyl-1-phosphono-propylamino)-succinicacid; or 3-(2-Naphthalen-2-yl-ethylcarbamoyl)-pentanedioic acid.
 10. Thepharmaceutical composition of claim 8, wherein the compound is3-(2-Biphenyl-4-yl-ethylcarbamoyl)-4-hydroxycarbamoyl-butvric acid. 11.The compound of claim 1, wherein A is


12. The compound of claim 1, wherein A is


13. The compound of claim 1, wherein A is


14. The compound of claim 1, wherein A is


15. The compound of claim 1 having the formula:


16. The pharmaceutical composition of claim 9, wherein the compound isfurther defined as3-[2-(4′-Cyano-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid.
 17. The pharmaceutical composition of claim 9, wherein thecompound is further defined as4-Hydroxycarbamoyl-3-[2-(4-pyridin-2-yl-phenyl)-ethylcarbamoyl]-butyricacid.
 18. The pharmaceutical composition of claim 9, wherein thecompound is further defined as4-Hydroxycarbamoyl-3-(4-phenyl-butylcarbamoyl)-butyric acid.
 19. Thepharmaceutical composition of claim 9, wherein the compound is furtherdefined as 4-Hydroxycarbamoyl-3-(2-phenoxy-ethylcarbamoyl)-butyric acid.20. The pharmaceutical composition of claim 9, wherein the compound isfurther defined as3-[2-(4′-Hydroxy-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid.
 21. The pharmaceutical composition of claim 9, wherein thecompound is further defined as3-(2,2-Diphenyl-ethylcarbamoyl)-4-hydroxycarbamoyl-butyric acid.
 22. Thepharmaceutical composition of claim 9, wherein the compound is furtherdefined as3-[2-(4′-Dimethylamino-biphenyl-4-yl)-ethylcarbamoyl]-4-hydroxycarbamoyl-butyricacid.
 23. The pharmaceutical composition of claim 9, wherein thecompound is further defined as4-Hydroxycarbamoyl-3-(5-hydroxy-pentylcarbamoyl)-butyric acid.
 24. Thepharmaceutical composition of claim 9, wherein the compound is furtherdefined as3-[(Biphenyl-4-ylmethyl)-carbamoyl]-4-hydroxycarbamoyl-butyric acid. 25.The pharmaceutical composition of claim 9, wherein the compound isfurther defined asN-[1-carboxy-2-(1H-indol-3-yl)-ethyl]-3-(3-phenyl-1-phosphono-propylamino)-succinicacid.
 26. The pharmaceutical composition of claim 9, wherein thecompound is further defined as3-(2-Naphthalen-2-yl-ethylcarbamoyl)-pentanedioic acid.